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Q: The gradlew signingReport for the SHA-1 key is not yielding any results - VS Code I've been trying to use ./gradlew signingReport to get the SHA-1 of my signing certificate but with no luck. I get this error:

> Task :app:signingReport
Variant: debug
Config: debug
Store: /home/user/.android/debug.keystore
Alias: AndroidDebugKey
Error: Missing keystore

I have created the project in VS Code on Ubuntu. Can someone help please?

A: Finally got it.
Once gradlew is installed on the system. Install the debug.keystore in the system's android directory and then move to the android project in the project and run this script. It works.


text:
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Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.
Funding: This work was supported by the Council of Scientific and Industrial Research, India grant 37(1535)/12/EMR-II. S.K.N. and A.C. acknowledge the receipt of an Institute Fellowship from Indian Institute of Technology Bhubaneswar. A.K.P. acknowledges ICMR, India (no. 45/25/2012-Bio/BMS) for providing a fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Exposure to temperatures beyond normal conditions or other stress factors may be lethal to cells. Thus, cells from several organisms have developed stress induced responses in order to guard themselves by the synthesis of a highly conserved set of proteins known as heat shock proteins (HSPs). An Italian scientist R. Ritossa observed the gene expression of puffing in the chromosomes of Drosophila melanogaster after exposure to heat which laid the pathway towards the discovery of HSPs . They are upregulated with an acute increase in temperature. Evidences reveal that these HSPs are essential for the survival of cells at both normal and elevated temperatures. Often they play a pivotal role in cellular homeostasis and stress response . Though the location of these proteins varies, mostly they are found in cytosol, nucleus, mitochondria and endoplasmic reticulum. Based on molecular weights, HSPs in archaea [3, 4], human , bacteria  and plants [5, 6] have been classified into five different categories, one of them is small heat shock protein family.
Small heat shock proteins (sHSPs) are ubiquitously expressed . They exhibit tissue specific expression in a variety of organisms with a molecular weight ~12–43 kDa . Most sHSPs are large molecular weight assemblies that have variable quaternary structure with a dynamic property which enable its subunits to freely exchange with oligomers. sHSPs usually possess a highly conserved "α-crystallin domain" at the centre, flanked by a C-terminal tail and preceded by a highly variable N-terminal region [9, 10]. Several studies have revealed that it is the "α-crystallin domain" in sHSPs that leads to the formation of large oligomeric assembly having oligomeric mass ranging between 200–800 kDa and is highly important for the subunit-subunit interactions [11–13]. sHSPs bind different client proteins under chemical and thermal stress and protect them from misfolding and aggregation. They also help in refolding of stressed proteins and are therefore known as molecular chaperones [14, 15].
Several small heat shock proteins have been reported to have increased chaperone function with increasing temperature [16–18]. This alteration in the chaperone function of small heat shock proteins is attributed to many factors, such as perturbation in oligomeric assembly, availability of hydrophobic patches, increased subunit exchange rate etc. For example, archaeal sHSPs (HSP16.5 from Methanococcus jannaschii and HSP26 from Saccharomyces cerevisiae), mammalian sHSPs (α-crystallin and HSP27 from human), plant sHSPs (HSP17.2 and HSP17.9 from sugarcane and HSP16.9 from wheat) and mycobacterial sHSPs (HSP16.3 from Mycobacterium tuberculosis) are some of the best characterised sHSPs, whose chaperone function was found to increase with increasing temperature [16–22]. Most of them have revealed that it is the dissociation of the oligomeric assembly of sHSPs (HSP26, α-crystallin, HSP17.2, HSP17.9 and HSP16.9) under environmental stress (especially enhanced temperature) that activates the chaperone function of these sHSPs [20, 21, 23]. In contrast, the enhanced chaperone function of HSP27 with increasing temperature has been attributed to the association of its oligomeric assembly . Contradictory to the above examples, some studies have also shown that with increasing temperature for certain sHSPs (HSP16.3 and HSP16.5), though an increase in chaperone function have been observed but its oligomeric size remained unaltered [16, 24]. The increased chaperone activity of such sHSPs with increasing temperature has not been due to any changes in its static oligomeric size but rather due to changes in dynamics of oligomeric assembly such as enhanced rate of subunit exchange [16, 24]. Besides the perturbation in oligomeric assembly caused due to temperature rise, a plethora of studies have also been investigated on the nature of forces that are employed by these sHSPs to bind the aggregation prone substrate proteins under thermal stress. Majority of these studies have shown hydrophobic interactions to be the driving force to bind the aggregation prone substrate proteins, which is a leading cause for the enhanced chaperone activity of sHSPs with increasing temperature [18, 25]. However, HSP16.3 and αB-crystallin are exceptions to such observations and exhibits decreased surface hydrophobicity with increasing temperature [16, 26]. It has been further shown that it is the electrostatic interaction rather than hydrophobic interaction playing a crucial role in the chaperone function of HSP16.3 and αB-crystallin [27, 28]. In summary, it is very much evident from the above instances that the static or dynamic properties of oligomeric assembly and nature of forces, governs the enhanced chaperone activity of sHSPs under thermal stress.
Recently, we have revealed that Mycobacterium leprae HSP18 (M. leprae HSP18), a class 3 heat shock protein, efficiently prevents the aggregation of substrate proteins under thermal and chemical stress . In that same paper, we have shown that M. leprae HSP18 exists in large oligomeric assembly (~29mer). It is basically an immunodominant antigen  which is specifically activated during intracellular growth and is involved in the survival of M. leprae pathogen within macrophages . It is believed that the molecular chaperone function of HSP18 may play a crucial role in the long term survival of M. leprae pathogen in infected hosts . But the mechanism behind the chaperone function of HSP18 is still not properly understood. Therefore, to understand the mechanism by which HSP18 prevents the aggregation of different client proteins from aggregation, we investigated the effects of heat on the conformation and chaperone activity of HSP18 using several biophysical and biochemical techniques. We found that heat stress alters the dynamics of oligomeric assembly and enhances the chaperone activity of M. leprae HSP18. Our results also provide the clue about the mode of interaction between HSP18 and the client proteins at elevated temperature.
Bovine insulin, citrate synthase (CS) from porcine heart, Dithiothreitol (DTT) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt (bis-ANS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Alexa fluor 350 and Alexa fluor 488 protein labeling kits (Catalog Nos. A-10170 and A-10235, respectively) were obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Malate dehydrogenase (MDH) from porcine heart, disodium hydrogen phosphate and mono sodium hydrogen phosphate were from Sisco Research Laboratories, India. All other chemicals were of analytical grade.
pQE31 expression vector harbouring HSP18 gene was obtained as a gift from Prof. Dharmalingham (Madurai Kamraj University, Madurai, India). Methods of expression and purification of wild-type M. leprae HSP18 has been described previously . Concentration of HSP18 was determined spectrophotometrically by measuring absorbance at 278 nm using extinction coefficient of 0.4 (mg/ml)-1cm-1. Concentration of this protein was also determined using Bradford assay. Molar concentration of HSP18 was calculated using its monomeric form. All biophysical assays were repeated with three different protein preparations.
Data are the mean ± standard deviation (SD) from triplicate determinations.
The chaperone activity of HSP18 in the absence or presence of NaCl was also determined using heat induced aggregation assay of citrate synthase (CS) at 43°C with the aid of same spectrophotometer . Briefly, HSP18 was pre-incubated with 0.05–0.5 M NaCl for 1 hr at 25°C. CS (0.06 mg/ml) aggregation was initiated by heating at 43°C in 50 mM phosphate buffer (pH 7.5) in the absence or presence of HSP18 pre-incubated without or with 0.05–0.5 M NaCl. The CS aggregation was monitored at 400 nm at 43°C for 1 hr in the kinetic mode using a data pitch of 10 seconds. Percent (%) protection for this aggregation assay was calculated using Eq 1. Data are the mean ± SD from triplicate determinations.
Thermal deactivation of malate dehydrogenase (MDH) was performed as previously described . Briefly, deactivation of 10 nM MDH in 50 mM phosphate buffer, pH 7.5 was initiated by heating the enzyme at 43°C in the absence or presence of 30 μM HSP18 pre-incubated with/without 0.05–0.5 M NaCl. MDH enzyme activity was assayed as described previously [29, 33] by collecting aliquots from the assay mixture incubated at 43°C for 10 mins. Experiments were also performed by replacing HSP18 with BSA to check specificity. Data are the mean ± SD from triplicate determinations.
Fluorescence of bis-ANS was measured as described previously  using Fluoromax 4P (Horiba Jobin Mayer, USA) spectrofluorimeter equipped with a thermostated cell holder. Fluorescence spectra of bis-ANS (10 μM) bound to HSP18 (0.05 mg/ml) were taken after incubation for 1 hr at 25, 31, 37 and 43°C. An excitation wavelength of 390 nm was used and the excitation and emission slits were 2.5 nm and 5 nm respectively. Fluorescence emission spectra were recorded in the range of 450–550 nm at the respective temperatures with a data pitch of 0.5 nm. The scan rate used for this assay was 240 nm/min. Each data point is the mean of triplicate measurements.
Tryptophan fluorescence spectra of HSP18 (3 μM) in 50 mM phosphate buffer, pH 7.5 were recorded from 310–400 nm at 25, 31, 37 and 43°C with a data pitch of 0.5 nm using spectrofluorometer (Fluoromax 4P, Horiba Jobin Mayer, USA) . The excitation wavelength was set to 295 nm. The scan rate used for this assay was 240 nm/min. Each data point is the mean of triplicate measurements.
Far- and near-UV CD spectra of HSP18 at different temperatures between 25–43°C were recorded using Chirascan CD spectropolarimeter (Applied Photophysics, Leatherhead, UK) equipped with peltier system as described previously . Protein concentrations used in far- and near-UV CD experiments were 0.2 mg/ml (in 10 mM phosphate buffer, pH 7.5) and 0.5 mg/ml (in 50 mM phosphate buffer, pH 7.5), respectively. Far-UV CD spectra were collected from 195–260 nm using a quartz cell with 1 mm path length, while the near-UV CD spectra were collected from 250–300 nm using 10 mm path length cell. Scan rate of 50 nm/min, data pitch of 0.5 nm, band width of 1 nm and response time of 0.2 seconds were used for far- and near- UV CD data collection. The reported spectra for far- and near-UV CD are the mean of five scans. For both experiments, spectra were recorded after incubating HSP18 at respective temperature for 1 hr. The curve-fitting program CONTINLL was used to analyze the secondary structural content of HSP18 at different temperatures .
M. leprae HSP18 (0.5 mg/ml, in 50 mM phosphate buffer, pH 7.5) was filtered through 0.22 μm disk membrane. Hydrodynamic radius of HSP18 oligomers pre-incubated without/with 0.5 M NaCl was measured by dynamic light scattering (DLS) employing a Zetasizer Nano S (Malvern Instruments, Malvern, UK) at 25, 31, 37 and 43°C with 1 hr incubation at each temperature prior to reading . Hydrodynamic radius of HSP18 oligomers was also measured at 25°C after pre-incubating the protein with 0.05, 0.15 and 0.5 M NaCl for 1 hr. During measurement, He–Ne laser (4 mW; 633 nm) was used and the detector angle was fixed at 173°. Every run composed of 12 acquisitions. Each data is an average of 5 such runs. RH was calculated of using the Stokes–Einstein equation: Eq 2 where, kB is Boltzmann's coefficient, T is absolute temperature, η is the viscosity of the medium and D is translational diffusion coefficient of the particles .
Oligomeric mass of HSP18 in the absence or presence of different NaCl concentrations (0.05, 0.15 and 0.5 M) was determined at 25°C using gel filtration chromatography as described previously . All these estimations were done in HPLC (Dionex, Sunnyvale, CA, USA) using an analytical gel filtration column (TSK-GEL G4000SWXL). Protein concentration used was 0.5 mg/ml in 50 mM phosphate buffer, pH 7.5. Calibration of the column was performed using different molecular weight standards: thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa) (Sigma Chemical Co.). Flow rate used for all measurements were 0.5 ml/min.
The structural stability of HSP18 was determined by both far-UV CD spectroscopy and differential scanning calorimetry (DSC) measurements.
The structural stability of HSP18 in the absence or presence of 0.5 M NaCl was determined using thermal denaturation experiments as described previously . Briefly, HSP18 (0.2 mg/ml in 10 mM phosphate buffer, pH 7.5) was heated from 25–80°C with a ramp rate of 0.5°C/min and the alteration in ellipticity at 222 nm was recorded using Chirascan CD spectropolarimeter (Applied Photophysics, Leatherhead, UK). Mid-point transition or melting temperature (Tm) was estimated using sigmoidal analysis as described previously  and van't Hoff equation was used to calculate the enthalpy change (ΔHvH) associated with the thermal transition of HSP18 in the absence and presence of 0.5 M NaCl.
The excess heat capacity was measured as a function of temperature in a VP DSC Microcalorimeter (GE Healthcare) to investigate thermal denaturation experiments of HSP18 in the absence or presence of NaCl. The samples were scanned from 20°C to 80°C with a scan speed of 60°C/hr at approximately 25 psi pressure. Prior to sample scan, the instrument was thermally stabilized by repeated buffer scans under similar conditions. Protein concentration used was 1 mg/ml in 50 mM phosphate buffer, pH 7.5 to obtain the melting profile of HSP18 in the absence or presence of NaCl. The thermograms obtained were analyzed using the in-built VPViewer software with Origin 7.0. The 2-state model of curve fitting was used to fit the raw thermograms.
Fluorescence conjugation of Alexa fluor 350 and Alexa fluor 488 fluorescent probes with purified recombinant M. leprae HSP18 was done as described previously [36, 37]. During tagging, we followed the procedure described by the manufacturer (Molecular Probes, Invitrogen, Carlsbad, CA, USA). Briefly, protein solutions (1 mg/ml) supplemented with 100 mM sodium bicarbonate (pH 7.2) were mixed with Alexa fluor probes for 1 hr at 25°C. BioGel P-30 column was used to separate the labeled protein from the unlabeled protein. The fraction, which contained the labeled HSP18, was collected and dialyzed against 50 mM phosphate buffer (pH 7.5) for 24 hr. The amount of HSP18 labeled with Alexa fluor 488 was estimated by measuring the absorbance at 278 and 494 nm of HSP18 and Alexa fluor 488, respectively. The degree of fluorescence labeling of Alexa fluor 350 to HSP18 was determined from the absorbance of HSP18 and Alexa fluor 350 at 278 and 346 nm, respectively. Concentrations of Alexa fluor 488 and Alexa fluor 350 were determined spectrophotometrically with the aid of molar extinction coefficients of 71,000 cm-1M-1 at 494 nm and 19,000 cm-1M-1 at 346 nm, respectively.
Fluorescence resonance energy transfer (FRET) technique was used to determine the rate of subunit exchange as described previously [36, 37]. For, subunit exchange experiments, Alexa fluor 350-labeled HSP18 acts as energy donor and Alexa fluor 488-labeled HSP18 acts as the energy acceptor. The proteins were incubated for 3 hr at different temperatures between 25–43°C in 50 mM phosphate buffer (pH 7.5) in a stoichiometric ratio of 1:1 (M/M). The reaction mixture was excited at 346 nm and the fluorescence emission spectra were taken from 400–600 nm at respective temperature using a Fluoromax-4P spectrofluorometer (Horiba Jobin Mayer, USA). The intensities at 440 nm and 513 nm were measured separately, which were used to calculate the subunit exchange rate constant (k) from the following equation: Eq 3 where Ft is the fluorescence intensity at 440 nm or 513 nm at different time intervals, Fo is the fluorescence intensity at 440 nm or 513 nm at t = 0, and k is the subunit exchange rate constant. Constants A and B were determined using conditions where A + B = 1 at t = 0 and A is the fluorescence intensity at t = ∞. Origin 8.0 software (OriginLab, Northampton, MA) was used to determine the subunit exchange rate constant of HSP18 subunits at various temperatures. We also performed the similar experiments in 50 mM phosphate buffer (pH 7.5) containing 0.05–0.5 M NaCl at 37°C.
All values are representative of means ± standard deviation of the number of experiments indicated in the respective figure legends. Student's t-test was used for the analysis of the statistical differences between the groups. Only those data were considered statistically significant, whose p-values were found to be ≤ 0.05.
M. leprae encounters various stresses such as hypoxia, nutrient depletion and oxidative stress during its survival in granulomata and macrophages of host species [38, 39]. Very little information exists in the literature regarding the factors which facilitate the virulence and survival of this pathogen under these stresses. Several antigenic proteins play critical role in the survival of M. leprae pathogen [40–42]. Some of these antigenic proteins having molecular weight 10–70 kDa, belong to heat shock protein family . Among these antigens, only 18 kDa antigen has been identified as small heat shock protein and termed as HSP18. Cole et al. reported the comparative analysis of M. leprae genome sequence with that of M. tuberculosis and revealed that, despite a massive gene decay in M. leprae, HSP18 gene has been retained , hinting over the fact that this small heat shock protein may be critical for virulence and survival of this pathogen. As M. leprae is non-culturable, the role of HSP18 in the survival of surrogate hosts (E. coli and M. smegmatis) carrying gene of this protein has been recently investigated under such stressed conditions where it has been found that the expression level of HSP18 increases . It has been also shown that HSP18 facilitated the multiplication of M. smegmatis within macrophages under conditions of nutrient depletion, hypoxia and oxidative stress . The expression level of HSP18 in recombinant E. coli has also increased moderately under heat stress . Such over-expression of HSP18 provides thermo-tolerance to E. coli which has been attributed to its chaperone function. However, the effect of heat on the chaperone function of HSP18 is still unclear. In view of the fact that, the structure and chaperone function of well known sHSPs are affected upon heating [16–20], we hypothesized that heat could alter the structure as well as chaperone function of HSP18.
To accomplish this, we first investigated the chaperone activity of M. leprae HSP18 at different temperatures. As the normal body temperature of human being, the host of pathogen M. leprae is ~37°C, the temperature range that has been selected for this study is 37 ± 6°C i.e. 31, 37 and 43°C. Apart from these three temperatures, room temperature (25°C) was also selected. Effect of heat/temperature on the chaperone function of HSP18 was assessed by monitoring the ability of HSP18 to prevent aggregation of insulin B chain. It was co-incubated with insulin at different temperatures (25, 31, 37 and 43°C) for 1 hr and DTT induced insulin aggregation assay was performed at the respective temperatures. At a ratio of 1:1 (w/w) of HSP18 to insulin, 24.6% protection against insulin aggregation was observed at 25°C (Fig 1A, trace 2 and 1B). Surprisingly, at the same chaperone to client protein ratio, the protection was increased to ~43% at 31°C (Fig 1A, trace 4 and 1B). When the temperature was further increased to 37°C, a further increase in the protection against insulin aggregation was observed (~68%) (Fig 1A, trace 6 and 1B). HSP18 protected substantially/almost the insulin aggregation (90%) at 43°C (Fig 1A, trace 8 and 1B). Therefore, it is quite evident from here that the temperature has profound influence on the aggregation prevention ability of HSP18. The chaperone function of HSP18 increases with increasing temperature.
Fig 1. Temperature-dependent chaperone-like activities of M. leprae HSP18.
(A) DTT-induced aggregation of 0.35 mg/ml insulin (Ins) in the absence and presence 0.35 mg/ml HSP18 at different temperatures (25, 31, 37 and 43°C). Aggregation was initiated by adding 20 mM DTT and scattering at 400 nm was monitored at the respective temperatures. Trace 1: Ins alone at 25°C; Trace 2: Ins + HSP18 at 25°C; Trace 3: Ins alone at 31°C; Trace 4: Ins + HSP18 at 31°C; Trace 5: Ins alone at 37°C; Trace 6: Ins + HSP18 at 37°C; Trace 7: Ins alone at 43°C; Trace 8: Ins + HSP18 at 43°C; (B) Percent protection ability of M. leprae HSP18 against insulin aggregation at different temperatures. Data are means ± the standard deviation from triplicate determinations. **p< 0.005 and ***p< 0.0005.
In order to understand whether the enhancement in the chaperone activity of HSP18 at elevated temperature is accompanied by any alteration in the proteins' conformations, secondary, tertiary and quaternary structure/conformation was studied at different temperatures (25, 31, 37 and 43°C). Far-UV CD spectra of HSP18 at these four temperatures are shown in Fig 2A, which clearly indicate that temperature has no effect on the secondary structure of HSP18. Data represented in Table 1 clearly indicated that HSP18 is a major β-sheet protein at 25°C. However, with increasing temperature, no change in the secondary structural elements of HSP18 was observed (Table 1). These data further confirmed that secondary structure of HSP18 was not altered upon the temperature rise from 25°C to 43°C. In addition, the effect of temperature/heat on the tertiary structure of HSP18 was studied using near-UV CD experiment (Fig 2B). The signal between 250–270 nm region arose from the five phenylalanine residues in HSP18 (Fig 2B). The signal beyond 270 nm corresponds to the single tyrosine and tryptophan residue in HSP18 (Fig 2B). When the near-UV CD spectra of HSP18 was recorded at elevated temperatures i.e. 31, 37 and 43°C, the spectral characteristics remained unaltered compared to that recorded at 25°C (Fig 2B). These data indicated that microenvironment of aromatic amino acid residues in HSP18 was not perturbed due to increase in temperature. The intrinsic tryptophan fluorescence spectra of HSP18 at different temperatures (25, 31, 37 and 43°C) agreed with the near-UV CD results (Fig 2C). No alteration in λmax as well as intensity of the intrinsic tryptophan fluorescence spectra was observed at higher temperature which further confirmed that temperature did not perturb the microenvironment of the single tryptophan residue (W33) of HSP18.
Fig 2. Effect of temperature on the structure of M. leprae HSP18.
Far-UV CD spectra (A) and near-UV spectra (B) of HSP18 at different temperatures (25, 31, 37 and 43°C). The concentrations of the protein samples used in far- and near-UV CD experiments were 0.2 and 0.5 mg/ml, respectively. (C) Tryptophan fluorescence spectra of HSP18 (0.05 mg/ml) were recorded from 310–400 nm at various temperatures (25, 31, 37 and 43°C). An excitation wavelength of 295 nm was used. Both the slit widths for excitation and emission were 5 nm. Data were collected at 0.5 nm wavelength resolution. (D) Intensity particle size distribution spectra of M. leprae HSP18 were recorded at various temperatures (25, 31, 37 and 43°C). Each of these spectra is an average of 48 scans. For each experiment, spectra were recorded after incubating HSP18 at respective temperature for 1 hr.
Table 1. Secondary structural elements of HSP18 at various temperatures.
Oligomeric structure of sHSPs is known to play a key role in its chaperone function. Several reports have revealed that increase in temperature have altered the oligomeric size of sHSPs [17, 21, 23]. Thus the oligomeric state of HSP18 was studied using dynamic light scattering (DLS) experiment between 25°C to 43°C. Fig 2D shows the intensity particle size distribution spectra of HSP18 at temperatures between 25–43°C. It was observed that at 25°C, the size distribution spectra of HSP18 was centred around 18.2 nm which corresponded to a molecular weight of 588 kDa. When HSP18 was incubated at higher temperatures (31, 37 and 43°C), no change in hydrodynamic radius was observed (Fig 2D). HSP18 could hold its native oligomeric size even at 43°C. This led us to conclude that temperature/heat did not perturb the oligomeric/quaternary structure of M. leprae HSP18. Overall, these findings revealed that the secondary, tertiary and quaternary structure of the protein remains unaltered in this temperature range.
The hallmark of sHSPs is to assemble into large oligomeric assembly of high molecular mass. Often subunits of different sHSPs exchange with each other by dynamic oligomeric dissociation/re-association which is pre-requisite for their chaperone function [16, 24, 37]. Since static oligomeric structure of HSP18 remained unaltered at elevated temperatures (31–43°C), we hypothesised that it is the changes in dynamics of oligomeric structure that caused enhanced chaperone function of HSP18 at these higher temperatures. First, we labelled HSP18 separately with Alexa fluor 350 (A-350) and Alexa flour 488 (A-488) and determined the degree of labeling. We observed that percentage labelling of Alexa fluor 350 and Alexa fluor 488 to HSP18 was 1:1 (M/M) which suggests that all proteins were labelled with these fluorophores. Size exclusion chromatographic experiments revealed that these labelling did not perturb the static oligomeric assembly and exhibited similar oligomeric mass/size compared to that of unlabelled HSP18 (S1 Fig). Then, we monitored the subunit exchange reaction at physiological temperature (37°C) by mixing an equimolar concentration of the donor, Alexa flour 350-labelled HSP18, with the acceptor, Alexa flour 488-labelled HSP18. Due to exchange between subunits of HSP18, we observed marked alteration in the donor and acceptor fluorescence (Fig 3A). Because of FRET, a time dependent decrease in fluorescence intensity at 440 nm for A-350 (labelled to HSP18) was observed with a concomitant time dependent increase in fluorescence intensity at 513 nm for A-488 (labelled to HSP18) which reached to its saturation in 3 hr at 37°C (Fig 4). To find out the rate constant (k) for the subunit exchange, the declining fluorescence intensity of A-350 (Fig 3B) and increasing fluorescence intensity of A-488 (Fig 3C) was fitted to the Eq 3. In both cases, we found that the rate constant (k) for the subunit exchange at 37°C was 0.039 min-1. This value of subunit exchange rate constant for HSP18 is almost comparable with the value of 0.0378 min-1 reported by Bova et al.  for the subunit exchange of αA-crystallin at 37°C. In the same paper, they also determined the rate constant for the subunit exchange of HSP27 at 37°C (0.0576 min-1) which is slightly higher compared to that of HSP18 and αA-crystallin. To understand the effect of temperature on the subunit exchange rate of HSP18, rate constant (k) of subunit exchange was also determined at 25, 31 and 43°C (Fig 3D). The subunit exchange rate constants for HSP18 at these three temperatures were 0.018, 0.023 and 0.149 min-1, respectively (Table 2 and Fig 3D). Thus, increasing the temperature from 25°C to 43°C, the magnitude of subunit exchange rate constant for HSP18 was increased almost 8 fold. Moreover, the subunit exchange rate constant was found to markedly increase (~4 fold) for HSP18 when the temperature was raised from 37°C to 43°C. Interestingly, almost similar rise in temperature (37°C to 42°C) also increased the magnitude of subunit exchange rate constant for αA-crystallin by ~4.2 fold . Moreover, we can say that HSP18 possess a dynamic quaternary structure even at 25°C and dynamics of oligomeric dissociation/re-association becomes rapid at elevated temperatures. In several previous studies, investigators demonstrated that the enhancement of the chaperone function of various sHSPs under elevated temperature is mostly associated with increased subunit exchange [16, 24, 47]. Therefore, we concluded that increased subunit exchange of HSP18 subunits at elevated temperature may be one of the basis for the enhancement of its chaperone function at higher temperature.
Fig 3. Determination of subunit exchange rate constant of HSP18 subunits at different temperatures.
(A) Time-course alterations in the emission spectrum of Alexa fluor-350 and 488 labeled M. leprae HSP18 due to subunit exchange at 37°C. The emission spectra were recorded at different time points after mixing equal amount of Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 (1 mg/ml each) at 37°C. The fluorescence spectra were recorded from 400 to 600 nm at 37°C using the excitation wavelength of 346 nm. The slit width of both excitation and emission monochromators was 5 nm each. The scan rate used for this assay was 240 nm/min. (B) Time-dependent decrease in fluorescence intensity at 440 nm for data shown in panel A. (C) Time-dependent increase in fluorescence intensity at 513 nm for data shown in panel A. (D) Temperature-dependent subunit exchange rate of M. leprae HSP18. Subunit exchange between Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 was monitored at 25, 31, 37 and 43°C. Symbols in panel B, C and D represent the experimental data points and the solid lines in these pannels represent the best fit of the data according to Eq 3.
Fig 4. bis-ANS binding to M. leprae HSP18.
(A) Fluorescence spectra of bis-ANS bound to HSP18 at different temperatures; (B) Fluorescence spectra of bis-ANS bound to HSP18 in the absence or presence of various concentrations of NaCl at 25°C. Protein concentration in all samples was 0.05 mg/ml and bis-ANS concentration was 10 M. The excitation wavelength was 390 nm. Excitation and emission slit widths were 2.5 and 5 nm, respectively. All the reported spectra were obtained by subtracting the respective control (bis-ANS alone) curves/spectra from the spectra of bis-ANS bound to HSP18.
Table 2. Subunit exchange rate constant of M. leprae HSP18 at different temperatures.
Apart from change in the kinetics of subunit exchange reaction, the alteration in the surface hydrophobicity of HSP18 at different temperatures was also determined. Generally, sHSPs possess several hydrophobic patches at the surface and these patches recognise and bind aggregation prone substrate proteins through hydrophobic interactions. It is usually believed that surface hydrophobicity governs the chaperone function of different sHSPs [48–51]. Additionally, several reports in the literature showed that the enhancement in the chaperone function of sHSPs is often accompanied by increased surface hydrophobicity [48, 49, 51]. The surface hydrophobicity of HSP18 at different temperatures (25, 31, 37 and 43°C) was estimated using bis-ANS. This hydrophobic probe is widely used for probing the hydrophobic sites of several sHSPs [48, 49, 51]. We observed that the fluorescence intensity of bis-ANS bound to HSP18 decreased marginally (~5%) during the rise in temperature from 25°C to 31°C (Fig 4A). We also noticed that the fluorescence intensity of bis-ANS bound to HSP18 exhibited gradual decrease (12% and 22%) upon heating at 37°C and 43°C, respectively (Fig 4A). All these data indicated that the availability of hydrophobic patches at the surface of HSP18 is reduced at elevated temperature. Thus, it seems that HSP18 is more hydrophobic at lower temperature and less hydrophobic at higher temperature. Also, we can conclude that hydrophobic interaction may not be the determinant factor behind the enhanced chaperone activity of HSP18 at higher temperature.
Failing to find a direct correlation between chaperone function and surface hydrophobicity in this temperature range prompted us to investigate some other forces which may be responsible for the enhanced chaperone function of HSP18 at higher temperature. In some of the sHSPs (α-crystallin and M. tuberculosis HSP16.3) it has been reported that, besides the hydrophobic interactions, the chaperone function of these sHSPs are also dependent on the electrostatic/ionic interactions [27, 28]. Charge-charge/electrostatic interactions are key to the substrate binding properties of these sHSPs. Therefore, to understand whether electrostatic interactions play a role in the chaperone function of M. leprae HSP18, we investigated the chaperone activity of HSP18 in the absence or presence of NaCl (0.05–0.5M) using DTT induced aggregation of insulin at 25°C. The effect of different concentrations of NaCl on the aggregation of insulin in the absence or presence of HSP18 is shown in Fig 5A. At 1:1.2 (w/w) ratio of insulin to HSP18, 49% protection against aggregation was observed in the absence of NaCl (Fig 5A, trace 2 and 5B). In the presence of 0.05 M NaCl, protection was reduced to ~42% (Fig 5A, trace 4 and 5B). As the NaCl concentration was increased from 0.05 M to 0.5 M, the protection ability of HSP18 was found to decrease further (~27%) (Fig 5A, trace 8 and 5B). The insulin aggregation assay was also carried out at 25°C by varying the ratio between HSP18 and insulin and 0.15 M NaCl. In both cases, the chaperone activity of HSP18 was found to decrease in the presence of 0.15 M NaCl (S2 Fig). To check, whether the effect is substrate specific or not, we examined the aggregation prevention ability of HSP18 in the absence and presence of NaCl (0.05–0.5 M) using another client protein i.e. citrate synthase (CS). Usually, CS tends to aggregate upon heating at 43°C. At 1:1.5 (w/w) ratio of CS to HSP18, 54% protection was observed in the absence of NaCl (Fig 5C, trace 2 and 5D). However, in the presence of 0.05 M NaCl, the protection ability was reduced to ~36% (Fig 5C, trace 4 and 5D). Upon further increase in NaCl concentrations (0.15 and 0.5 M), the protection ability was further reduced (27% and 21%, respectively) (Fig 5C, traces 6 & 8 and 5D). Thus from both the aggregation assays it was inferred that HSP18 exhibits reduced chaperone activity in the presence of NaCl. Besides aggregation prevention ability, HSP18 can also prevent the thermal inactivation of enzyme [malate dehydrogenase (MDH)] . To understand whether the weakening of electrostatic interactions impair this ability of HSP18, the thermal deactivation of MDH was carried out without or with different HSP18 protein samples (pre-incubated without or with 0.05–0.5 M NaCl concentrations) (Fig 6). MDH alone could retain only 39% of its activity. However, in the presence of 30 μM HSP18, it could retain ~63% enzyme activity (Fig 6). When HSP18 was pre-incubated with 0.05–0.5 M NaCl and then subjected to MDH thermal deactivation assay, the loss of MDH activity was increased and it could only retain ~57% (at 0.05 M NaCl) to ~46% (at 0.5 M NaCl) enzyme activity (Fig 6). So as to check the specificity, parallel experiments were also carried out where HSP18 was replaced with BSA (Fig 6), but, BSA failed to exhibit thermal deactivation prevention ability both in the absence or presence of 0.05–0.5 M NaCl (Fig 6). Appropriate controls were also done to check the enzymatic activity of MDH in the presence of various concentrations of NaCl without HSP18. Thus it was inferred that the thermal deactivation prevention ability of HSP18 was reduced in the presence of NaCl. Besides, the effect of NaCl on the surface hydrophobicity of HSP18 was also monitored using bis-ANS experiment. Interestingly, with varying concentrations of NaCl ranging from 0–0.5 M, the fluorescence intensity of bis-ANS bound to HSP18 was found to increase by ~19% at 25°C (Fig 4B), suggesting over the fact that surface hydrophobicity of HSP18 was enhanced in the presence of NaCl. However, the chaperone function of HSP18 was reduced in the presence of NaCl at the same temperature. Therefore, it can be concluded from these evidences that electrostatic interactions play a crucial role for proper execution of chaperone function by M. leprae HSP18 at 25°C.
Fig 5. Effect of NaCl on the chaperone activity of M. leprae HSP18.
DTT-induced aggregation of 0.35 mg/ml insulin at 25°C (panel A) and thermal aggregation of 0.06 mg/ml CS at 43°C (panel C) in the absence or presence of different HSP18 samples. Both insulin and citrate synthase are denoted as client proteins. Trace 1: Client protein (CP) alone; Trace 2: CP +HSP18; Trace 3: CP +0.05 M NaCl; Trace 4: CP + HSP18 + 0.05 M NaCl; Trace 5: CP +0.15 M NaCl; Trace 6: CP + HSP18+0.15 M NaCl; Trace 7: CP +0.5 M NaCl; Trace 8: CP + HSP18+0.5 M NaCl. Each data point is the average of triplicate measurements. The percent protection ability of different HSP18 samples against insulin and CS aggregation are presented in panels B and D, respectively. The insulin: HSP18 ratio was 1:1.2 (w/w) and the CS: HSP18 ratio was 1:1.5 (w/w). Data are means ± standard deviation from triplicate determinations. NS = Not significant, *p< 0.05, **p< 0.005 and ***p< 0.0005.
Fig 6. Effect of NaCl on the thermal deactivation prevention ability of M. leprae HSP18.
The MDH enzyme activity was measured in the absence and presence of 30 μM HSP18 pre-incubated in the absence or presence of 0.05–0.5 M NaCl while it was thermally denatured at 43°C. Similar assays were also performed with 30 μM BSA alone or pre-incubated with 0.05–0.5 M NaCl. Data are means ± standard deviation from triplicate determinations. *p< 0.05, **p< 0.005.
We also checked the chaperone activity of HSP18 at physiological temperature (37°C) in the absence and presence of different NaCl concentrations (0.15 and 0.5 M) using DTT induced insulin aggregation assay. In the presence of 0.15 M NaCl, the chaperone function of HSP18 was found to decrease by ~17.5% at 37°C (Fig 7B and 7C), while the same was found to decrease by ~11.6% at 25°C (Fig 7A and 7C). The more dramatic effect was observed in the presence 0.5 M NaCl. At this NaCl concentration, we noted that the chaperone function of HSP18 was lowered by ~21.6% at 25°C (Fig 7A and 7C). However, at the same salt concentration, the protection ability of HSP18 was reduced significantly (~45%) at 37°C (Fig 7B and 7C). We also found the greater reduction in the chaperone activity of HSP18 in the presence of 0.15 M and 0.5 M NaCl at other two higher temperatures (31°C and 43°C) (data not shown). Together, our data revealed that the interactions between HSP18 and chemically stressed client protein were weakened greatly in the presence of NaCl at higher temperature, which is the cause for reduction in the chaperone function of HSP18. Possibly, electrostatic interactions govern the recognition and binding of aggregation prone substrate proteins by HSP18 at normal as well as elevated temperatures.
Fig 7. Effect of temperature on the chaperone activity of M. leprae HSP18 in the presence of NaCl.
The aggregation of insulin B chains (0.35 mg/ml) initiated by the addition of DTT (20 mM) was used to determine the chaperone activity of HSP18 (0.42 mg/ml) at (A) 25°C and (B) 37°C. (C) Percentage protection of insulin (0.35 mg/ml) aggregation by HSP18 in the absence or presence of various NaCl concentrations at two different temperatures. Data are means ± the standard deviation from triplicate determinations. *p< 0.05, **p< 0.005 and ***p< 0.0005.
Once it was concluded that electrostatic interactions are important for the chaperone function of M. leprae HSP18, we were keen to know the molecular basis for the decreased chaperone function of HSP18 when the electrostatic forces were weakened by addition of NaCl. Electrostatic interactions are noncovalent bonding interactions, either attractive or repulsive in nature. It includes coulombic interactions and more sequence specific interactions like ion pairing or hydrogen bonding . Electrostatic interaction is known to play key role in protein stability and function [53, 54]. It has long been implicated in maintaining thermal stability of thermophilic proteins [55, 56]. Valle et al. reported that Ca2+ ion induced decreased thermal stability of α-crystallin caused mainly due to electrostatic interaction between Ca2+/α-crystallin which eventually led to its decreased chaperone functionality . So, the structural stability of HSP18 in the absence and presence of 0.5 M NaCl was compared against thermal stress using far-UV CD measurements. The change in the ellipticity magnitude at 222 nm was monitored over a temperature range from 25°C to 80°C. Then, the fraction unfolded (αU) for both systems were calculated using the following equation: Eq 4 where θF represents ellipticity value measured at 25°C for the native protein, θt represents ellipticity value measured at any temperature and θU represents ellipticity value measured at 80°C for the unfolded protein. As shown in Fig 8A, the thermal denaturation profiles of both proteins (HSP18 in the absence and presence of 0.5 M NaCl) were sigmoidal in nature. Although formation of an intermediate state cannot be negated, a two state transition model exhibited a significantly good fitting. Sigmoidal analysis of far-UV CD profiles at 222 nm demonstrated that HSP18 in absence of any NaCl underwent thermal unfolding with a midpoint transition or melting temperature (Tm) of 60.9°C (Table 3). In the presence of 0.5 M NaCl, the Tm value shifted to 54°C (Fig 8A and Table 3). The decrease in Tm value (~6.9°C) clearly suggested that the presence of NaCl significantly reduced the structural stability of HSP18. As thermal denaturation profiles of HSP18 in the absence and presence of 0.5 M NaCl gave a significantly good fit with the two state transition model, the change in enthalpy (∆HvH) of this thermal transition was evaluated using van't Hoff equation: Eq 5 where Keq(folded→unfolded) = αU/(1 - αU). The value of van't Hoff enthalpy (ΔHvH) associated with the thermal transition of HSP18 (in absence of NaCl) was ~122.5 kJ/mol, which decreased to 92.2 kJ/mol for HSP18 in the presence of 0.5 M NaCl (Fig 8B and Table 3) which further confirmed that weakening of electrostatic interactions lowered the structural stability of HSP18 under thermal stress. The reversibility of this unfolding process of HSP18 in the absence or presence of 0.5 M NaCl was also checked by reheating the protein sample immediately after cooling at 25°C from the previous scan. It is worthwhile to mention that the Tm and ΔHvH values so obtained from the reheating process were similar to that of during first heating. Therefore, it was inferred that the unfolding of HSP18 in the absence and presence of 0.5 M NaCl is reversible in nature.
Fig 8. Effect of electrostatic interaction on the structural stability of HSP18.
The thermal stability of HSP18 in the absence or presence of 0.5 M NaCl was determined using far-UV CD spectroscopy (panel A) and differential scanning calorimetry (panel C). The experimental data points (mentioned by symbols in panel A and sold lines in panel C) were fitted according to two-state model and the solid lines (in panel A) and dotted lines (in panel C) represent the best fit. (B) The data represented in panel A was fitted to Eq 5 in order to obtain the values of van't Hoff enthalpy (∆HvH) HSP18 in the absence or presence of 0.5 M NaCl.
Table 3. Thermodynamic parameters for thermal unfolding of HSP18 in the absence and presence of 0.5 M NaCl obtained from far UV-CD and DSC measurements.
The effect of NaCl on the structural stability of HSP18 was further assessed by differential scanning calorimetry (DSC). We also performed this experiment to understand the presence of any intermediate during the thermal unfolding of HSP18 in the absence and presence of 0.5 M NaCl. DSC experiments revealed sharp transition during the unfolding of HSP18 in the absence and presence of 0.5 M NaCl (Fig 8C). Also, alterations in the melting temperature (Tm) and van't Hoff enthalpy changes (ΔHvH) were observed in the presence of 0.5 M NaCl (Fig 8C and Table 3). While HSP18 in absence of NaCl exhibited a Tm and ΔHvH of ~62.1°C and ~ 135.9 kJ/mole, respectively, the magnitude of Tm and ΔHvH associated with the unfolding of HSP18 in the presence of 0.5 M NaCl were reduced (~54.5°C and 121 kJ/mole, respectively). These results were in agreement with our thermal denaturation experiments which were carried out using far-UV CD spectroscopy (Fig 8A and Table 3). Moreover, the unfolding process in DSC measurements produced narrow as well as relatively symmetric peak for HSP18 in the absence or presence of 0.5 M NaCl (Fig 8C) which indicated that both unfolding transitions may be two-state, reversible and highly cooperative . Further verification of two step behavior was done by comparing the calorimetric (ΔHcal) and van't Hoff enthalpy changes (ΔHvH) as obtained from DSC measurements. The ΔHcal and ΔHvH magnitudes were found to be almost equal for the unfolding of HSP18 in the absence and presence of 0.5 M NaCl (Table 3). These results further confirmed that the denaturation process of M. leprae HSP18 (in the absence and presence of 0.5 M NaCl) could be accurately approximated by a two step model and rules out the possibility for the existence of an intermediate state . As in both the systems, the existence of only two states were confirmed i.e. native state and the fully denatured state, the denaturation process of HSP18 in the absence or presence of 0.5 M NaCl could be termed as cooperative unfolding process. Reversibility of both the denaturation process was also checked by scanning to the thermal transition maximum, cooling to 20°C and scanning again upto 80°C. The Tm, ΔHcal and ΔHvH values so obtained from the rescan remained almost unchanged. Thus these results again revealed that the thermal denaturation process of M. leprae HSP18 in the absence or presence of 0.5 M NaCl is a reversible, cooperative unfolding process. Previous studies on various sHSPs suggested that structural stability modulated their chaperone function [29, 48, 60]. Moreover, some studies revealed that enhancement in the chaperone activity of various sHSPs are accompanied by increased structural stability [29, 48, 60]. Thus it can be inferred from these data that weakening of the electrostatic interactions led to a decrease in the structural stability of HSP18, which reduced the chaperone activity of this protein.
To investigate whether the decreased structural stability of the protein due to the weakening of the electrostatic interactions, is accompanied by alteration in its static oligomeric assembly, the oligomaric size/mass of HSP18 was estimated in the absence and presence of 0.05–0.5 M NaCl at 25°C using gel filtration chromatography. The native HSP18 (in absence of NaCl) was eluted with a single peak at 9.05 ml (Fig 9A, peak 1). The peak position of HSP18 elution was shifted to 9.15 ml, 9.61 ml and 10.55 ml in the presence of 0.05 M, 0.15 M and 0.5 M NaCl, respectively (Fig 9A, peaks 2–4) which indicated the dissociation of static oligomeric assembly of HSP18 in the presence of NaCl. To quantify the oligomeric mass corresponding to these elution volumes, a standard curve was generated with several molecular weight standards (Fig 9A, inset). The oligomeric mass of HSP18 in absence of NaCl was found to be 563.6 kDa which is in well agreement with our previous estimation  In contrast, the oligomeric mass of HSP18 in the presence of 0.05 M, 0.15 M and 0.5 M NaCl was found to be 514, 335.5 and 223.7 kDa, respectively. Altogether, these data suggest that the electrostatic interactions led to the reduction in the oligomeric size of HSP18, consisting of 29 subunits in absence of NaCl to approximately 12 subunits in the presence of 0.5 M NaCl.
Fig 9. Effect of NaCl on the oligomeric mass/size of M. leprae HSP18.
(A) Gel filtration profile of HSP18 in the absence or presence of 0.05–0.5 M NaCl at 25°C. TSK-GEL G4000SWXL column (7.8 mm x 30 cm; 5 μm) was first equilibrated with 50 mM phosphate buffer (pH 7.5) with or without 0.05–0.5 M NaCl. Subsequently, 50 μl of HSP18, pre-incubated without or with 0.05–0.5 M NaCl was injected into the column. The flow rate used in this experiment was 0.5 ml/min. The oligomeric mass of different HSP18 samples was estimated using the standard curve (inset). (B) Intensity particle size distribution spectra of M. leprae HSP18 in the absence or presence of 0.05–0.5 M NaCl at 25°C. (C) Intensity particle size distribution spectra of M. leprae HSP18 in the presence of 0.5 M NaCl at different temperatures (25, 31, 37 and 43°C). Each spectrum is an average of 48 scans. HSP18 was incubated at respective temperatures in the absence or presence of 0.05–0.5 M NaCl for 1 hr prior to reading. Protein concentration used was 0.5 mg/ml in 50 mM phosphate buffer, pH 7.5.
To confirm this fact, we also estimated the hydrodynamic radius of HSP18 in the absence or presence of 0.05–0.5 M NaCl using dynamic light scattering experiment at 25°C. This study clearly ascertained our gel filtration chromatographic results and showed that static oligomeric size of M. leprae HSP18 was reduced with increasing concentrations of NaCl (Fig 9B). In absence of NaCl, the observed hydrodynamic radius of M. leprae HSP18 was ~18.2 nm, which was reduced upto ~17.4 nm in the presence of 0.05 M NaCl. When NaCl concentrations were raised to 0.15 and 0.5 M, the observed hydrodynamic radius of M. leprae HSP18 was further reduced and found to be centered around ~16.5 and ~14.9 nm, respectively (Fig 9B). Thus a net decrease of ~3.3 nm in the hydrodynamic radius of M. leprae HSP18 was observed in the presence of 0.5 M NaCl at 25°C. We also explored how the static oligomeric size of HSP18 is altered in the presence of 0.5 M NaCl at higher temperatures (31, 37 and 43°C). As gel filtration chromatography is usually carried out at room temperature, and not at higher temperatures because of possible interaction between column matrix and protein, dynamic light scattering experiments were carried for this particular investigation. We observed that the magnitude of hydrodynamic radius of HSP18 decreased further with increasing temperatures (Fig 9C). While at 25°C, the hydrodynamic radius of HSP18 in the presence of 0.5 M NaCl was~14.9 nm, the same was found to be reduced to ~13.6 and ~12.8 nm at 31°C and 37°C respectively (Fig 9C). Further reduction in its hydrodynamic radius (~11.2 nm) was observed when the temperature was raised to 43°C, thereby resulting a net decrease of ~3.7 nm in the hydrodynamic radius of M. leprae HSP18 in the presence of 0.5 M NaCl (Fig 9C). This additional reduction of static oligomeric size of HSP18 at higher temperature in the presence of 0.5 M NaCl may be responsible for the further decrease in the chaperone function of 0.5 M NaCl incubated HSP18 (Figs 5 and 6). Previously, several studies demonstrated that reduced structural stability of sHSPs is often associated with dissociation of their static oligomeric assembly [61, 62]. These perturbations often reduced the chaperone function of these sHSPs [61, 62]. Recently, our group has also shown that a point mutation in the "α-crystallin domain" of M. leprae HSP18 caused its structural destabilization and dissociation of its oligomeric assembly . The same study also revealed that such alteration in the quarternary structure and stability of the HSP18 was accompanied by reduced chaperone function. Therefore, we can conclude that weakening of electrostatic interactions in M. leprae HSP18 led to the oligomeric dissociation and structural destabilization which perhaps reduced its chaperone function. The reduced chaperone function of M. leprae HSP18 at elevated temperatures in the presence of NaCl was likely due to the reduction in its static oligomeric size, hinting over the fact that static oligomeric assembly is pre-reqisite for the chaperone function of M. leprae HSP18.
Once it was evidenced that weakening of electrostatic interactions caused dissociation of static oligomeric assembly of HSP18, we were keenly interested to understand how the dynamics of oligomeric assembly of HSP18 are altered upon weakening of electrostatic interactions. We already demonstrated that the rate of subunit exchange is increased upon raising the temperature from 25°C to 43°C (Fig 3D) and may therefore play an important role in the enhancement of chaperone function of M. leprae HSP18 at elevated temperatures. We also demonstrated that weakening of electrostatic interactions resulted in the diminished chaperone activity of M. leprae HSP18 (Figs 5 and 6). Therefore, we hypothesized that kinetics of subunit exchange of HSP18 subunits may have retarded upon weakening of electrostatic interactions. To test this particular hypothesis, we executed the subunit exchange studies (by FRET) at different salt concentrations (0–0.5 M NaCl) at 37°C. In absence of NaCl, the rate constant for the subunit exchange of HSP18 subunit was 0.039 min-1 at 37°C (Fig 10, Table 4). In contrast, at the same temperature, the magnitude of the same was decreased in the presence of 0.05 M NaCl (to 0.031 min-1) (Fig 10, Table 4). Upon further increase in NaCl concentrations (0.15 and 0.5 M), the subunit exchange rate constant of M. leprae HSP18 subunits was found to be reduced further (0.028 min-1 and 0.024 min-1, respectively) (Fig 10, Table 4). The rate constant value of subunit exchange of HSP18 subunit was also reduced in the presence of 0.5 M NaCl at 25°C (S1 Table). Thus it is evident from here that dynamics of subunit exchange of M. leprae HSP18 is lowered when its electrostatic interactions are weakened. This leads to assume that the subunits of HSP18 interact with each other through electrostatic interactions and possibly the inter/intrapolypeptide interactions within the HSP oligomer affect/alter upon weakening of electrostatic interaction. Perhaps rearrangements of the subunits are essential for the binding of stressed client proteins and preventing their aggregation. Exhibition of similar trends in the subunit exchange phenomenon and chaperone function by M. leprae HSP18 clearly indicates that the dynamics of oligomeric assembly of M. leprae HSP18 are one of the determinants for the chaperone function of this protein at room temperature (25°C) as well as higher temperatures (31–43°C).
Fig 10. Effect of NaCl on subunit exchange rate of M. leprae HSP18.
Subunit exchange between Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 was monitored at 37°C in the absence or presence of 0.05–0.5 M NaCl. The concentration of both labeled protein was 1 mg/ml. The fluorescence spectra (400–600 nm) were recorded at various time intervals for different samples at 37°C. The excitation wavelength was 346 nm. In order to find out the subunit rate constant (k) of HSP18 in the absence or presence of 0.05–0.5 M NaCl, the curves were fitted according to the Eq 3.
Table 4. Subunit exchange rate constant of M. leprae HSP18 in the absence or presence of 0.05–0.5 M NaCl at 37°C.
In summary, the present study has demonstrated that HSP18 has a dynamic quaternary structure at room temperature (25°C) and apart from this unique feature, electrostatic interaction plays a crucial role in proper exhibition of chaperone function at this temperature. We also demonstrated that the subunit exchange rate of HSP18 subunits increases at different higher temperatures, resulting in increased chaperone function of HSP18. We successfully revealed that weakening of electrostatic interactions reduces the static oligomeric size, dynamics of oligomeric assembly and structural stability of HSP18. Altogether, these alterations resulted drastic decrease in its chaperone function at elevated temperatures. A possible model figure which reflects the schematic mechanism for the chaperone function of M. leprae HSP18 at various temperatures is shown in Fig 11. As HSP18 could prevent the insulin aggregation at physiological temperature (37°C) more efficiently, it implies that this protein can bind various aggregation prone client proteins in vivo. Previously, we demonstrated that HSP18 can prevent the thermal killing of E.coli at higher temperature . Perhaps, enhanced chaperone activity of HSP18 at elevated temperature may be responsible for this prevention. Overall, the subunit exchange and electrostatic interactions play important role in the chaperone function of M. leprae HSP18. But, how the chaperone function of M. leprae HSP18 is modulated under various non thermal stresses such as hypoxia, nutrient depletion, oxidative stress etc., needs to be explored in details. These studies would improve our understanding on the molecular basis behind the chaperone function of M. leprae HSP18.
Fig 11. A proposed model which reflects the schematic mechanism for the chaperone function of M. leprae HSP18 at various temperatures.
S1 Fig. Effect of fluorophore labeling on the oligomeric mass of M. leprae HSP18 at 25°C.
Gel filtration profile of HSP18 unlabelled or labelled with A-350/A-488 at 25°C. TSK-GEL G4000SWXL column (7.8 mm x 30 cm, 5 μm) was equilibrated with 50 mM phosphate buffer, pH 7.5. Subsequently, 50 μL of HSP18, unlabelled/labelled with A-350/A-488 was injected into the column. Flow rate used was 0.5 ml/min. Profiles has been normalised to 0–1 scale.
S2 Fig. Chaperone activity with varying concentration of HSP18 in the presence of 0.15 M NaCl at 25°C.
Percentage protection by HSP18 against DTT-induced insulin aggregation at 25°C. The ratio of client protein to HSP18 (w/w) was 1:1.2, 1:1.5 and 1:1.8, respectively. Data are means ± standard deviation from triplicate determinations. *p< 0.05 and **p< 0.005.
S1 Table. Subunit exchange rate constant of M. leprae HSP18 in the absence or presence of 0.5 M NaCl at 25°C.
We thank Prof. K. P. Das and Dr. Victor Banerjee, Bose Institute, Kolkata, India for their useful suggestions during DLS experiments. We also thank Mr. Dipak C. Konar, Bose Institute, Kolkata, India for collecting DLS data. S.K.N. and A.C. acknowledges the receipt of an Institute Fellowship from Indian Institute of Technology Bhubaneswar. A.K.P. acknowledges ICMR, India (no. 45/25/2012-Bio/BMS) for providing fellowship.
Conceived and designed the experiments: SKN AB. Performed the experiments: SKN AKP SSR AC. Analyzed the data: SKN AB. Contributed reagents/materials/analysis tools: AB. Wrote the paper: AB SKN.
1. Ritossa F. A new puffing pattern induced by heat shock and DNP in Drosophila. Experientia. 1962;18:571–3.
3. Trent JD. A review of acquired thermotolerance, heat-shock proteins and molecular chaperones in Archaea. Fems Microbiol Rev. 1996;18:249–58.
4. Kim KK, Yokota H, Santoso S, Lerner D, Kim R, Kim SH. Purification, crystallization, and preliminary X-ray crystallographic data analysis of small heat shock protein homolog from Methanococcus jannaschii, a hyperthermophile. J Struct Biol. 1998;121(1):76–80. Epub 1998/05/09. doi: S1047-8477(98)93969-7 [pii] pmid:9573624.
5. Schlesinger MJ. Heat shock proteins. J Biol Chem. 1990;265(21):12111–4. Epub 1990/07/25. pmid:2197269.
6. Kotak S, Larkindale J, Lee U, von Koskull-Doring P, Vierling E, Scharf KD. Complexity of the heat stress response in plants. Curr Opin Plant Biol. 2007;10(3):310–6. Epub 2007/05/08. doi: S1369-5266(07)00046-5 [pii] pmid:17482504.
7. Boston RS, Viitanen PV, Vierling E. Molecular chaperones and protein folding in plants. Plant Mol Biol. 1996;32(1–2):191–222. Epub 1996/10/01. pmid:8980480.
8. Sharma KK, Santhoshkumar P. Lens aging: effects of crystallins. Biochim Biophys Acta. 2009;1790(10):1095–108. pmid:19463898; PubMed Central PMCID: PMC2743770.
9. Plesofsky-Vig N, Vig J, Brambl R. Phylogeny of the alpha-crystallin-related heat-shock proteins. J Mol Evol. 1992;35(6):537–45. Epub 1992/12/01. pmid:1474606.
10. Waters ER. The molecular evolution of the small heat-shock proteins in plants. Genetics. 1995;141(2):785–95. Epub 1995/10/01. pmid:8647410; PubMed Central PMCID: PMC1206773.
11. Berengian AR, Bova MP, McHaourab HS. Structure and function of the conserved domain in alphaA-crystallin. Site-directed spin labeling identifies a beta-strand located near a subunit interface. Biochemistry. 1997;36(33):9951–7. Epub 1997/08/19. bi9712347 [pii]. pmid:9296605.
12. Koteiche HA, Berengian AR, McHaourab HS. Identification of protein folding patterns using site-directed spin labeling. Structural characterization of a beta-sheet and putative substrate binding regions in the conserved domain of alpha A-crystallin. Biochemistry. 1998;37(37):12681–8. Epub 1998/09/16. bi9814078 [pii]. pmid:9737844.
13. Berengian AR, Parfenova M, McHaourab HS. Site-directed spin labeling study of subunit interactions in the alpha-crystallin domain of small heat-shock proteins. Comparison of the oligomer symmetry in alphaA-crystallin, HSP 27, and HSP 16.3. J Biol Chem. 1999;274(10):6305–14. Epub 1999/02/26. pmid:10037719.
14. Hartl FU. Molecular chaperones in cellular protein folding. Nature. 1996;381(6583):571–9. Epub 1996/06/13. pmid:8637592.
15. Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992;355(6355):33–45. Epub 1992/01/02. pmid:1731198.
16. Fu X, Chang Z. Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis. Biochem Biophys Res Commun. 2004;316(2):291–9. Epub 2004/03/17. [pii]. pmid:15020216.
17. Lelj-Garolla B, Mauk AG. Self-association and chaperone activity of Hsp27 are thermally activated. J Biol Chem. 2006;281(12):8169–74. Epub 2006/01/27. doi: M512553200 [pii] pmid:16436384.
18. Das KP, Surewicz WK. Temperature-induced exposure of hydrophobic surfaces and its effect on the chaperone activity of alpha-crystallin. FEBS Lett. 1995;369(2–3):321–5. Epub 1995/08/07. doi: 0014-5793(95)00775-5 [pii]. pmid:7649280.
19. Kim DR, Lee I, Ha SC, Kim KK. Activation mechanism of HSP16.5 from Methanococcus jannaschii. Biochem Biophys Res Commun. 2003;307(4):991–8. Epub 2003/07/25. doi: S0006291X03013020 [pii]. pmid:12878210.
20. Haslbeck M, Walke S, Stromer T, Ehrnsperger M, White HE, Chen S, et al. Hsp26: a temperature-regulated chaperone. EMBO J. 1999;18(23):6744–51. Epub 1999/12/03. pmid:10581247; PubMed Central PMCID: PMC1171736.
21. Tiroli-Cepeda AO, Ramos CH. Heat causes oligomeric disassembly and increases the chaperone activity of small heat shock proteins from sugarcane. Plant Physiol Biochem. 2010;48(2–3):108–16. Epub 2010/02/09. S0981-9428(10)00007-0 [pii]. pmid:20137963.
22. van Montfort RL, Basha E, Friedrich KL, Slingsby C, Vierling E. Crystal structure and assembly of a eukaryotic small heat shock protein. Nat Struct Biol. 2001;8(12):1025–30. Epub 2001/11/10. nsb722 [pii]. pmid:11702068.
23. Chebotareva NA, Kurganov BI, Muranov KO, Asryants RA, Ostrovsky MA. Role of thermoinduced dissociation in interaction between alpha-crystallin as an oligomeric chaperone and glyceraldehyde-3-phosphate dehydrogenase as an oligomeric protein substrate. Dokl Biochem Biophys. 2009;428:245–8. Epub 2010/09/21. pmid:20848910.
24. Bova MP, Huang Q, Ding L, Horwitz J. Subunit exchange, conformational stability, and chaperone-like function of the small heat shock protein 16.5 from Methanococcus jannaschii. J Biol Chem. 2002;277(41):38468–75. Epub 2002/08/15. M205594200 [pii]. pmid:12176992.
25. Lee GJ, Roseman AM, Saibil HR, Vierling E. A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J. 1997;16(3):659–71. Epub 1997/02/03. pmid:9034347; PubMed Central PMCID: PMC1169668.
26. Reddy GB, Das KP, Petrash JM, Surewicz WK. Temperature-dependent chaperone activity and structural properties of human alphaA- and alphaB-crystallins. J Biol Chem. 2000;275(7):4565–70. Epub 2000/02/15. pmid:10671481.
27. Mao Q, Ke D, Chang Z. Electrostatic interactions play a critical role in Mycobacterium tuberculosis Hsp16.3 binding of substrate proteins. Biochemistry (Mosc). 2001;66(8):904–8. Epub 2001/09/22. doi: BCM66081111 [pii]. pmid:11566061.
28. Khalili-Hezarjaribi H, Yousefi R, Moosavi-Movahedi AA. Effect of temperature and ionic strength on structure and chaperone activity of glycated and non-glycated alpha-crystallins. Protein Pept Lett. 2012;19(4):450–7. Epub 2011/09/22. doi: BSP/ PPL/ E pub/0401 [pii]. pmid:21933131.
29. Nandi SK, Rehna EA, Panda AK, Shiburaj S, Dharmalingam K, Biswas A. A S52P mutation in the 'alpha-crystallin domain' of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function. FEBS J. 2013;280(23):5994–6009. Epub 2013/09/13. pmid:24024660.
30. Booth RJ, Williams DL, Moudgil KD, Noonan LC, Grandison PM, McKee JJ, et al. Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria. Infect Immun. 1993;61(4):1509–15. Epub 1993/04/01. pmid:8454357; PubMed Central PMCID: PMC281393.
31. Dellagostin OA, Esposito G, Eales LJ, Dale JW, McFadden J. Activity of mycobacterial promoters during intracellular and extracellular growth. Microbiology. 1995;141 (Pt 8):1785–92. Epub 1995/08/01. pmid:7551043.
32. Lini N, Rehna EA, Shiburaj S, Maheshwari JJ, Shankernarayan NP, Dharmalingam K. Functional characterization of a small heat shock protein from Mycobacterium leprae. BMC Microbiol. 2008;8:208. Epub 2008/12/02. [pii]. pmid:19040732; PubMed Central PMCID: PMC2629775.
33. Nandi SK, Chakraborty A, Panda AK, Sinha Ray S, Kar RK, Bhunia A, et al. Interaction of ATP with a Small Heat Shock Protein from Mycobacterium leprae: Effect on Its Structure and Function. PLoS Negl Trop Dis. 2015;9(3):e0003661. Epub 2015/03/27. [pii]. pmid:25811190; PubMed Central PMCID: PMC4374918.
34. Banerjee V, Das KP. Interaction of silver nanoparticles with proteins: a characteristic protein concentration dependent profile of SPR signal. Colloids Surf B Biointerfaces. 2013;111:71–9. Epub 2013/06/25. doi: S0927-7765(13)00317-2 [pii] pmid:23792543.
35. Banerjee V, Das KP. Modulation of pathway of insulin fibrillation by a small molecule helix inducer 2,2,2-trifluoroethanol. Colloids Surf B Biointerfaces. 2012;92:142–50. Epub 2011/12/20. [pii]. pmid:22178183.
36. Biswas A, Goshe J, Miller A, Santhoshkumar P, Luckey C, Bhat MB, et al. Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin. Biochemistry. 2007;46(5):1117–27. Epub 2007/01/31. pmid:17260942.
37. Biswas A, Das KP. Differential recognition of natural and nonnatural substrate by molecular chaperone alpha-crystallin-A subunit exchange study. Biopolymers. 2007;85(2):189–97. Epub 2006/11/15. pmid:17103422.
38. Cosma CL, Sherman DR, Ramakrishnan L. The secret lives of the pathogenic mycobacteria. Annu Rev Microbiol. 2003;57:641–76. Epub 2003/10/07. pmid:14527294.
39. Cunningham AF, Spreadbury CL. Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog. J Bacteriol. 1998;180(4):801–8. Epub 1998/02/24. pmid:9473032; PubMed Central PMCID: PMC106957.
40. Mehra V, Bloom BR, Bajardi AC, Grisso CL, Sieling PA, Alland D, et al. A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein. J Exp Med. 1992;175(1):275–84. Epub 1992/01/01. pmid:1730920; PubMed Central PMCID: PMC2119087.
41. Singh I, Yadav AR, Mohanty KK, Katoch K, Bisht D, Sharma P, et al. Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy. Cell Immunol. 2012;278(1–2):63–75. Epub 2012/11/06. doi: S0008-8749(12)00123-2 [pii] pmid:23121977.
42. Pessolani MC, Smith DR, Rivoire B, McCormick J, Hefta SA, Cole ST, et al. Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae. J Exp Med. 1994;180(1):319–27. Epub 1994/07/01. pmid:8006590; PubMed Central PMCID: PMC2191570.
43. Vega-Lopez F, Stoker NG, Locniskar MF, Dockrell HM, Grant KA, McAdam KP. Recognition of mycobacterial antigens by sera from patients with leprosy. J Clin Microbiol. 1988;26(12):2474–9. Epub 1988/12/01. pmid:3068245; PubMed Central PMCID: PMC266928.
44. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, et al. Massive gene decay in the leprosy bacillus. Nature. 2001;409(6823):1007–11. Epub 2001/03/10. pmid:11234002.
45. Maheshwari JJ, Dharmalingam K. Protective role of Mycobacterium leprae small heat-shock protein in heterologous hosts, Escherichia coli and Mycobacterium smegmatis, grown under stress. J Med Microbiol. 2013;62(Pt 7):959–67. Epub 2013/04/13. [pii]. pmid:23579398.
46. Bova MP, McHaourab HS, Han Y, Fung BK. Subunit exchange of small heat shock proteins. Analysis of oligomer formation of alphaA-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations. J Biol Chem. 2000;275(2):1035–42. Epub 2000/01/08. pmid:10625643.
47. Bova MP, Ding LL, Horwitz J, Fung BK. Subunit exchange of alphaA-crystallin. J Biol Chem. 1997;272(47):29511–7. Epub 1997/12/31. pmid:9368012.
48. Biswas A, Das KP. Role of ATP on the interaction of alpha-crystallin with its substrates and its implications for the molecular chaperone function. J Biol Chem. 2004;279(41):42648–57. Epub 2004/08/05. M404444200 [pii]. pmid:15292216.
49. Nahomi RB, Huang R, Nandi SK, Wang B, Padmanabha S, Santhoshkumar P, et al. Acetylation of lysine 92 improves the chaperone and anti-apoptotic activities of human alphaB-crystallin. Biochemistry. 2013;52(45):8126–38. Epub 2013/10/17. pmid:24128140; PubMed Central PMCID: PMC3889837.
50. Chowdary TK, Raman B, Ramakrishna T, Rao CM. Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity. Biochem J. 2004;381(Pt 2):379–87. Epub 2004/03/20. BJ20031958 [pii]. pmid:15030316; PubMed Central PMCID: PMC1133843.
51. Smykal P, Masin J, Hrdy I, Konopasek I, Zarsky V. Chaperone activity of tobacco HSP18, a small heat-shock protein, is inhibited by ATP. Plant J. 2000;23(6):703–13. Epub 2000/09/21. doi: tpj837 [pii]. pmid:10998182.
52. Kauzmann W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1969;14(-):1–69.
53. Nakamura H. Roles of electrostatic interaction in proteins. Q Rev Biophys. 1996;29(1):1–90. Epub 1996/02/01. pmid:8783394.
54. Matthew JB. Electrostatic effects in proteins. Annu Rev Biophys Biophys Chem. 1985;14:387–417. Epub 1985/01/01. pmid:3890885.
55. Petsko GA. Structural basis of thermostability in hyperthermophilic proteins, or "there's more than one way to skin a cat". Methods Enzymol. 2001;334:469–78. Epub 2001/06/12. doi: S0076-6879(01)34486-5 [pii]. pmid:11398484.
56. Szilagyi A, Zavodszky P. Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure. 2000;8(5):493–504. Epub 2000/05/10. doi: S0969212600001337 [pii]. pmid:10801491.
57. del Valle LJ, Escribano C, Perez JJ, Garriga P. Calcium-induced decrease of the thermal stability and chaperone activity of alpha-crystallin. Biochim Biophys Acta. 2002;1601(1):100–9. Epub 2002/11/14. doi: S1570963902004296 [pii]. pmid:12429508.
58. Bruylants G, Wouters J, Michaux C. Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design. Curr Med Chem. 2005;12(17):2011–20. Epub 2005/08/17. pmid:16101501.
59. Marky LA, Breslauer KJ. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers. 1987;26(9):1601–20. Epub 1987/09/01. pmid:3663875.
60. Nagaraj RH, Panda AK, Shanthakumar S, Santhoshkumar P, Pasupuleti N, Wang B, et al. Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics. PLoS One. 2012;7(1):e30257. Epub 2012/01/25. PONE-D-11-22108 [pii]. pmid:22272318; PubMed Central PMCID: PMC3260246.
61. Kundu M, Sen PC, Das KP. Structure, stability, and chaperone function of alphaA-crystallin: role of N-terminal region. Biopolymers. 2007;86(3):177–92. Epub 2007/03/09. pmid:17345631.
62. Stromer T, Fischer E, Richter K, Haslbeck M, Buchner J. Analysis of the regulation of the molecular chaperone Hsp26 by temperature-induced dissociation: the N-terminal domail is important for oligomer assembly and the binding of unfolding proteins. J Biol Chem. 2004;279(12):11222–8. Epub 2004/01/15. M310149200 [pii]. pmid:14722093.
Is the Subject Area "Hydrodynamics" applicable to this article?


text:
Taproot Online
The Ohio State University at Newark – Volume 8, 2012
Amber Dingess
Duane Gray
Elizabeth Byers Gaubert
Kayla Liston
McKenzie L. Shaw
Miguel Johnson
Retha Carolyn Murray
Tabatha Baughman
Victoria Chanfrau
Breana Jossey
Daphne Hicks-Blankenship
Shirley Curtis
Steven Foley
William G. Brown
Beth Goodwin
J. Kelley Anderson
Kory McGaughey
An Unsuccessful Death
Ripple Explorers
Three Dark Fantasies
Abby Spear
Alex Carroll
Darcy Heller
Jared Frick
Jen Struckman
Jenneca Vaughn
Jennifer Lusetti
Laurinda Johnson
Michael Reimer
Heidi Smith
Norita Yoder
TAKE BACK THE STREETS . . . FOR REAL
Winner, 2012 Promising Writer Award
As an eighteen year old girl, when men come from the shadows shooting at your friends, that is the first thought that crosses your mind. Right? Mine too. And my mom's.
It is the late 1980's, back in the days of 3.2% beer that eighteen year olds can legally drink. Nancy Snyder, not yet my mother, is barhopping downtown with her friends in an area of Columbus, Ohio called the Short North. Tonight is a rare break from the chaos of the AIDS epidemic; Nancy's nickname is Lady Death because of her natural immunity to the mystery virus. She carries bleeding, dying men from bed to deathbed day in and day out because hospitals are too afraid to take them in. The stench of death clings to her clothes, but the smell of sweat and weak alcohol cover up some of it for the evening. Her parents do not know where she is . . . she knows they are better off ignorant. They will probably never fully accept that their only daughter is a lesbian. But for now, it is how it is, and this night is an escape.
Being in the dingiest part of town, where graffiti covers every surface and gangs run amok in the darkness, Nancy fits in with other leather-clad rebels. Everything is better until she leaves the bar, only to watch some acquaintances shot down steps ahead of her. The blood stench she barely escaped is back now, filling the air in no less sickly a manner than it ever does around the ill. The police do not even bother showing up. That night Nancy and the rest of the growing LGBT (lesbian, gay, bisexual, transgendered) community decided to meet more often; time to take back the streets!
All too often, once-beautiful cities fall into ruin as industry leaves and workers flock to suburbia. The Short North, like other central cities in the United States, experienced a boom and bust before falling into forgotten piles of rubble. Many of these inner city neighborhoods, including those of Chicago, Illinois, where my mother was born, are taken under the wing of the LGBT community and rebuilt into glorious meccas of art and high society. The galleries, refurbished homes, and rainbow flag-sailing street corners of the Short North today scream GAY!, but the city, and even WOSU, the local public media station broadcasting from the OSU campus, purposefully leave out the LGBT people entirely from the rich history of the city. In a personal email to me, my mom reveals details of the transformation that usually are swept under the rug. She urges me to look back at the history of the city and read between the lines.
Several gay bars dotted the downtown and Short North area. In 1978, what would become the Metropolitan Community Church (MCC) of Columbus started meeting in the Twilight Lounge, a gay show bar on High Street in the Short North when the area was still rough. Later MCC moved to a storefront on High Street just north of 5th Avenue, and eventually to St. Paul's on Broad, where I went to church when I was little. But this was before St. Paul's allowed an LGBT church to move in. In 1980, the year my mom graduated from high school, MCC was still on High Street. After Mom and her friends joined the "Take Back the Streets" movement, she started cleaning up graffiti while the burly-looking queens stood around as guards. The Blue Berets patrolled the streets at night in groups of three or four. Everyone carried a walkie-talkie to notify police at a moment's notice of gang activity encroaching upon the clean-up effort. The numerous boarded-up houses were scrubbed clean and they picked up bag upon bag of trash. Gay men started moving into the area and fixing up houses, their new immaculate flowerbeds flowing down to the sidewalks in rainbow bursts of color. Cleanliness and a sense of community pushed the gangs aside and drug activity fizzled out. Businesses started moving in; the tide had turned.
This changing point in the city's history is painfully easy to explain, yet so many people remain oblivious because no one bothers to explain it. Some, like gun enthusiast Jeff Garvas, only touch on this time period in their writing because of groups like the all-but-forgotten Pink Pistons. Like the Blue Berets, this gay-straight alliance gun club helped gays defend themselves against gay bashers and street gangs. The Pink Pistons motto is "Armed gays don't get bashed!" and they still exist today as an advocacy group. Garvas points out that the rate of assault against homosexuals here in Columbus soars above the national rate year after year. Maybe this is a result of the ignorance that has been allowed to continue for decades.
A day after I read her email, I sat down with my mom and her partner to watch a DVD documentary called Columbus Neighborhoods: Short North. They were both excited because mom's partner, Ann, is a professor at The Ohio State University and WOSU has won awards for its making of this new documentary. My parents were sure that the huge part of the Short North's transformation that they both lived through would be on the DVD and they hoped I could learn more about it than what their combined memories could teach me. Before pressing play, I skimmed the back of the case. It claimed that I would see a "vibrant, exciting" neighborhood . . . including "the tale of the arches, the rise – and abrupt fall – of the Columbus Union Station, an incredible reunion of Civil War soldiers, the circus magnate who mesmerized a town, and the emergence of local festivals and traditions . . . " and then FINALLY I saw " . . . a story of how a grass roots movement transforms a run-down, forgotten neighborhood into Columbus' center for arts and culture." Now that is what I want to see! Snuggled on the couch with our cat, the three of us watched with anticipation through slides of Victorian Village, Italian Village, Harrison West, and Fly Town. They interviewed gallery owners, including my friend Hayley's mom Sherrie, but the interviews looked a little off. How did they manage to interview only straight gallery owners? There are – what? – maybe two straight gallery owners in the entire arts district. As the documentary wrapped up with beautiful shots of the city today, my poor mom stomped off in outrage yelling, "Where were we?" and Ann trailed behind her whispering something about them including gays once somewhere in there. Yes, I noticed, gays were mentioned once–as being scary.
Later, watching the film for a second and third time at my apartment, I never was able to put my finger on the grassroots movement mentioned on the case. I popped up onto the Short North website to see if things have been corrected by now. After all, the film is from 2010. Maybe historians showed up to right the wrongs sometime in the past year.
Whoa! Surprise, surprise. The history page of http://www.shortnorth.org includes Union Station, Arch City, Goodale Park, and the Sells Brothers Circus. And that's all folks! The grand Union Station was built in 1850 when the city had its industrial boom. The arches of the station inspired the architecture of what is known today as The Cap, a strip of expensive restaurants and bars. Arch City became the Short North's national nickname in 1888 when Columbus was chosen to host a centennial celebration for the creation of the Northwest Territory. Columbus put up arches to light the streets, but they disappeared by 1916. Today the arches are back, and at night they light up the sky like a mile long rainbow. Goodale Park, a gift from Dr. Lincoln Goodale in 1851, is now the home of Comfest. Finally, the Sells Brothers Circus sold shares to Barnum and Bailey and eventually became part of the Ringling Brothers Greatest Show on Earth in 1911. These are the highlights of Short North history. These apparently tell the story Columbus wants the public to hear. These events are the important ones.
The Ohio State University as a public school needs to worry about what the "public" thinks about them. Maybe Columbus and WOSU leave out gays from the history of the city because they think they are taking a necessary precaution. What if OSU were suddenly openly advocating for gay rights on a large scale? Sure, they have clubs and support groups. They do AIDS testing and hand out condoms on the campus. But outside of the "diversity" bubble necessary for any large school, OSU and WOSU do not want to take a stand. The best way to avoid controversy is to ignore whatever is the issue. OSU does not want to be an advocate any more than they want to be anti-gay. They want to be accepting in the most politically safe sense.
Obviously none of these people have taken a stroll up High Street before. None of these smart documentary folks have really visited the Short North. Everyone acknowledges the time in between the "good" parts, but no one can quite place their finger on how the city grew back into itself. No one knows why the Short North is even more glorious now. I think it was that grassroots movement so briefly mentioned. I think they should have interviewed the survivors. Do they even know how few survivors there are? Most of them are female because so many men fell victim to AIDS, but those females remember what happened. To them, the AIDS epidemic was as disastrous as the Holocaust was for us Jews. To them, leaving the rebuilding of the city and the symbolic rebuilding of the gay community out of our communal history is as tragic as forgetting about the Holocaust. Or even forgetting World War II entirely! Just ask my mom, the lesbian Jew of Polish descent.
A social group is unpopular with the government. They have no rights, they are discriminated against, and they face prejudices and persecution. One has recognition decades later after nearly being extinct; the other still cheers for every small win after they too were nearly wiped out. Will the government recognize the freedom to love? Will we college students, the future leaders, even learn about the injustices going on under our noses? If no one bothers to interview Nancy Snyder or any of the other survivors, will there be anything to recognize once they are all gone? Anything to learn from? No, there will only be what we are taught right now and what we teach in the future. Take Back the Streets and "Armed gays don't get bashed!" will disappear with the deep symbolism of the rainbow flag that flies all over the Short North.
My mother's memories will die with her some day and our public records will never care. The gay community in Columbus will seem to have popped up out of nowhere because their rich, colorful, dangerous history is not to be found. Not even at OSU, the largest college campus in the country.
Heidi Smith is a first-year honors student at Ohio State Newark, majoring in social work. She writes: "Growing up, I was never sure why other kids thought I was different. Having two moms didn't make me underprivileged, nor did it make me particularly lucky. It wasn't until I was really discriminated against in high school that it hit me; I am different. My same-sex parents give me a different perspective. The piece I wrote is my way of speaking out on their behalf. My parents are not worse than any other parents! In fact, they might be better because their "marriage" is strong. I will never be able to thank them enough for loving me, motivating me, and always being proud of me. I am proud of them too and I will not stop fighting for their basic rights."


The David Nicholson Mares' Hurdle is yet another Grade 1 race on the first day of the Cheltenham Festival. Open to mares aged four or older, the Mares' Hurdle is run over two miles, four furlongs and has nine hurdles along the way.
In the 10 previous renewals of this race, Willie Mullins has trained all but two of the winners (Quevega accounts for six of those wins) but it's the 2017 champion, Apple's Jade for Gordon Elliott, who is at the head of the market.
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Free bets, enhanced odds and other offers for the Mares' Hurdle will appear as soon as the bookies release them, but check out the latest Cheltenham Festival betting offers for the other Festival promotions and bonuses currently available.
<New Customers can get Benie Des Dieux at an enhanced 33/1 to win Mares Hurdle. Max Stake £1. Winnings paid in free bets. Full T&Cs Apply. 18+|Gamble Aware.
The Mares' Hurdle is one of the newest races at the Cheltenham Festival. It's very quickly reached Grade 1 status and is therefore a well-regarded race with a big prize fund. A major reason for its growth in stature was Quevega's incredible run of wins.
She won the Mares' Hurdle an amazing six times which required a remarkable feat of training from Willie Mullins. Only twice has Mullins failed to win the Mares' Hurdle and he has another great chance in 2019 in the shape of firm favourite, Benie Des Dieux.
It is great to see so many high class mares competing in the Champion Hurdle at this year's Cheltenham Festival. Apple's Jade, Laurina and Verdana Blue will all prove to be quality additions to Tuesday's championship race but it has hurt the quality of the field for the Mares' Hurdle.
Some pundits have gone as far as to suggest that tighter qualification criteria for the Mares' Hurdle should be introduced to make it the competitive contest for mares that it was supposed to be but that debate is for another day. Punters on Tuesday will only be concerned with who is going to win the 2019 Mares' Hurdle.
On the face of it, it is not a tough call for punters to make. Benie Des Dieux won this race 12 months ago when finishing a couple of lengths ahead of Apple's Jade and Ruby Walsh has described her as his best prospect of the entire Cheltenham Festival.
The only real worry is that she is yet to have a run this season but connections are not concerned. She has more class than any of her competition and could have been competitive in a couple of different races at Cheltenham so this is one favourite who is expected to oblige punters at 10/11 with RaceBets.
Connections of most horses in the field for the Mares' Hurdle has a tough decision to make. Lady Buttons' enthusiastic owners had many discussions with her trainer, Philip Kirby, about whether she should go for the Mares' Hurdle or instead have a crack at the Queen Mother Champion Chase.
They only decided to race on Tuesday at the 11th hour but may be rewarded for their choice. Lady Buttons was a very popular winner of the Grade 2 Yorkshire Rose Mares' Hurdle at Doncaster last time out which marked a return to hurdles after some improved performances over fences.
Chasing may be the longer term plan for Lady Buttons but there's more than enough to like about her hurdling ability over the 2 mile 4 furlong trip of the Mares' Hurdle. She also proven to be versatile in terms of the going which is highly important given the poor weather in the forecast for the Cheltenham area so Lady Buttons looks the main danger to Benie Des Dieux at 10/1 with Ladbrokes.
2008 – Whiteoak – jockey Jason Maguire, trainer Donald McCain, Jr.
As one of the newer additions to the Cheltenham Festival the Mares' Hurdle is one of those races which punters are still trying to get their heads around. The 2m4f race is a top class contest which always attracts entries from some of the best mares around but profiling a winner is no easy task for a few reasons.
The most obvious of these is the dominance of Quevega in the race's early history. Willie Mullins' wonder mare did great things for the anticipation and calibre of the Mares' Hurdle but her six wins have had a major impact on the stats which is something punters must be very aware of.
The trends will show you that a high percentage of the Mares' Hurdle winners were sent off as the favourite. Look a little deeper and you'll see that Quevega was the favourite for all six of her wins and that winning favourites have been much rarer since she left the stage.
Similarly, Quevega was 10 when she won her sixth Mares' Hurdle but generally speaking this is a race for younger horses with six and seven-year-olds particularly favoured.
It's a little different with the record of trainers though. In addition to his six wins with Quevega, Willie Mullins' vice like grip of the race was further strengthened with wins from Glens Melody, Vroum Vroum Mag and Benie Des Dieux. You can take nothing away from the feat of training required to get any of those horses prepared to win the Mares' Hurdle but talk of the dominance of Irish trainers should be taken into context as Gordon Elliott was the only victorious other Irish trainer in the first 11 renewals of the race.
It is worth noting that between 2009 and 2018 there were well over 100 British runners but just 10 claimed even a place.
Having been added to the festival offering in 2008, this contest is one of the newer events to appear over the four days. Originally taking place on the final day, the race was moved to its current opening day slot the year after. Despite its relative youth this 2m4f contest has already featured its fair share of scintillating performances and drama.
Initially known as the David Nicholson Mares' Hurdle, the race is still officially registered under this title despite the sponsorship provided by OLBG from 2011. David Nicholson certainly earned his right to have a festival contest named in his honour given the remarkable success he enjoyed at the jewel in jumps racing's crown. The man who affectionately came to be known as "The Duke" won five festival races as a jockey and 17 as a trainer.
The inaugural running of the race was won by a short head in a thrilling finish by Donald McCain Jr's five-year-old Whiteoak. The race was taken by a five-year-old again in 2009 but this time the race was over a long way before the finish line, as Willie Mullins' Quevega sauntered to a 14 length success. Little did we know at the time but this was just the beginning of a record breaking sequence for this superstar mare.
Nothing could touch this daughter of Robin Des Champs from 2009-2014 as she rattled in six consecutive wins in this race. When collaring stablemate Glens Melody close home in 2014, she surpassed the Cheltenham Festival record of the legendary Golden Miller who won five consecutive Gold Cups in the 1930's. On the back of Quevega's heroics the race was upgraded to Grade 1 status in 2015 having previously been a Grade 2 contest.
Quevega was retired in 2014 but the Mullins dominance was set to continue into 2015. The 2015 Mares Hurdle will live long in the memory of both punters and bookmakers. Following a stellar opening day at the festival for supporters of Willie Mullins, as his hotpots, Douvan, Un de Sceaux and Faugheen all won, there was a fortune running on to the heavily favoured Annie Power in this race.
The bookies hid behind their satchels as Annie Power strode away from the field approaching the final fence… only to completely misjudge it and crash to the turf. A fall which it is estimated saved the bookies around £40 million. It was 2014 second Glens Melody who was left to pick up the pieces and provide Mullins with his seventh win in the first eight editions of this race. Mullins followed up in 2016 with yet another win, this time with Vroum Vroum Mag.
In 2017 we had something remarkable though: a winner not trained by Willie Mullins, as Apple's Jade romped home for Gordon Elliott. Mullins restored the natural order for the race the following year though as he saddled Benie Des Dieux to success in 2018, giving him his ninth win from just 11 renewals!