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Title: Tipping the Scale from Disorder to Alpha-helix: Folding of Amphiphilic Peptides in the Presence of Macroscopic and Molecular Interfaces}
Article:
Abstract:
Secondary amphiphilicity is inherent to the secondary structural elements of proteins. By forming energetically favorable contacts with each other these amphiphilic building blocks give rise to the formation of a tertiary structure. Small proteins and peptides, on the other hand, are usually too short to form multiple structural elements and cannot stabilize them internally. Therefore, these molecules are often found to be structurally ambiguous up to the point of a large degree of intrinsic disorder in solution. Consequently, their conformational preference is particularly susceptible to environmental conditions such as pH, salts, or presence of interfaces. In this study we use molecular dynamics simulations to analyze the conformational behavior of two synthetic peptides, LKKLLKLLKKLLKL( LK) and EAALAEALAEALAE( EALA), with built-in secondary amphiphilicity upon forming an alpha-helix. We use these model peptides to systematically study their aggregation and the influence of macroscopic and molecular interfaces on their conformational preferences. We show that the peptides are neither random coils in bulk water nor fully formed alpha helices, but adopt multiple conformations and secondary structure elements with short lifetimes. These provide a basis for conformation-selection and population-shift upon environmental changes. Differences in these peptides’ response to macroscopic and molecular interfaces( presented by an aggregation partner) can be linked to their inherent alpha-helical tendencies in bulk water. We find that the peptides’ aggregation behavior is also strongly affected by presence or absence of an interface, and rather subtly depends on their surface charge and hydrophobicity.
Introduction:
The partitioning, folding and aggregation properties of soluble peptides and small proteins at hydrophobic/hydrophilic interfaces( HHI) play a crucial role in biomedically important processes. One example that has been extensively investigated over the last years are amyloid-fibril forming peptides which are responsible for many human disorders, among them severe neurodegenerative diseases[1–3]. The amphiphilic character of the involved amyloidogenic proteins and peptides, such as islet amyloid polypeptide( IAPP), amyloid-β( Aβ) peptide or α-synuclein is responsible both for their aggregation propensity as well as for their strong interface activity. For several of the amyloidogenic peptides not only interaction with biomembrane surfaces but also membrane penetrating, membrane rupturing, and pore-forming mechanisms are discussed[4]. This aspect of partitioning and penetration through hydrophobic barriers like cell membranes links them to a related class of natural and synthetic peptides, so called cell penetrating peptides( CPP). They have gained importance as a tool for the targeted delivery of non-permeable therapeutics like other peptides, proteins, oligonucleotides or nanoparticles into the cell.[5, 6] The folding and aggregation equilibria of these systems are usually complex and very sensitive to external factors( ion strength, pH, etc.). In particular, folding patterns are very diverse, spanning from different helical structures to various β-structure-rich arrangements[4], and the transitions between these states are coupled to changes in peptide/protein concentration, the presence of initial aggregates as seeds or the presence of membranes, interfaces or surfaces. Aggregate formation appears to occur both in the α-helical and in the β-sheet state, and in several systems a partially helical structure has been reported prior to fibril formation, while the fibrils themselves typically show β-structure-rich regions[4, 7]. For many of these systems an intrinsically disordered state has been found at low concentrations in solution, and it is speculated that this state is an essential intermediate and of great importance for self assembly and aggregation[8]. In particular, very little is known about the low concentration species that are crucial for the early stages of aggregation and that are folding/refolding intermediates. Their molecular state is largely unknown since they are very difficult to detect and characterize experimentally at low concentrations. In addition, their ill-defined fold and their highly dynamic behavior with conformations that remain stable only for short timescales contribute to the challenges in detection. Yet these states are of incredibly high interest as they may well be the actually toxic species in neurodegenerative diseases. Due to the amphiphilic nature of the peptides/proteins, folding into more defined conformations is observed at interfaces such as the air/water interface or at a biomembrane. In addition to this stabilization of conformations, the interface propensity also leads to an increase in local peptide/protein concentration. Both factors presumably further promote aggregation and fibril formation( see[9–12]). The effect of the interface on the folding process of these—in bulk mostly disordered—peptides/proteins and their aggregation is not understood yet in its entirety[13–16]. Molecular simulation can shed light on the above-described disordered, half-folded, and highly-dynamic states at low concentration. This information can be used to interpret ensemble data from spectroscopic experiments such as NMR, IR or other. In addition, a mechanistic understanding of intrinsically disordered states and the driving forces that trigger folding and aggregation can be obtained. Atomistic insight into unfolded or partially folded intermediates can help to identify the role of different contributions, such as polar and hydrophobic interactions, hydrogen bonding, conformational propensities of amino acid sequences, etc. In the present study, we use classical atomistic molecular dynamics simulations to investigate the environment-dependent folding equilibrium of two( synthetic) model peptide systems—with a special focus on the influence of soft hydrophobic interfaces and oligomer formation on this equilibrium. The two chosen systems—a cationic model peptide based on LK repeats[17] and EALA, a pH-switchable anionic peptide based on the so-called GALA system[18]—are both designed to display secondary amphiphilicity in the α-helical conformation( Fig 1). Both systems are reported to have an affinity to biomembranes and to oligomerize in solution( and in/on the membranes)[17–19]. Peptides composed of leucine and lysine residues have been analyzed in recent studies for their adsorption behavior on hard surfaces with enhanced sampling methods[20], as well as for their role as templates for biomineralization via coarse grained simulations[21]. As we will demonstrate in the course of this paper, both systems display a highly dynamic conformational behavior, with a sizeable amount of intrinsic disorder in solution and an ability to form α-helices under certain conditions. Thus, our synthetic peptides share the key features of their conformational behavior with natural systems such as α-synuclein. With their highly repetitive designed sequences they are good model systems to analyze the contribution of different interactions and driving forces to the coupled equilibria of folding, aggregation and partitioning at( macroscopic) hydrophobic/hydrophilic interfaces( see Fig 2), and to characterize typical microscopic intermediates and pathways. The significant motifs in biological systems such as α-synuclein may not be as regular as in the model systems but the general features( such as repeats of charged and hydrophobic elements—see S1 Fig) are comparable. In the present study we have chosen the most simple representation of a HHI, the( air) vacuum/water interface. Experimentally, the air/water interface is used to study the folding equilibria of various protein systems, and its importance for amyloidogenic intrinsically disordered proteins such as A-β or α-synuclein has recently been recognized[9, 11, 22, 23]. Air/water interfaces are abundantly present in many experimental systems due to agitation of the samples and they appear to affect fibril formation and growth. From a computational point of view the vacuum/water interface is an ideal soft hydrophobic/hydrophilic model interface that allows to systematically study generic effects and fundamental driving forces. In the course of the paper we will first characterize the folding equilibrium of individual LKKLLKLLKKLLKL( LK) and EAALAEALAEALAE( EALA) molecules in bulk water( equilibrium A in Fig 2), in particular with respect to potential intrinsic disorder of the individual molecule in bulk, i. e. without a further ordering environmental effect. Then we will subsequently study, how mesoscopic interfaces( in the form of the air/water interface; indicated as connecting arc D in Fig 2), as well as nanoscale interfaces( in the form of a second molecule, as a representative of early stages of oligomerization; indicated as connecting arc E in Fig 2) influence the folding equilibrium of the two molecules. As a last step we will show, how the presence of a HHI affects aggregate formation and possibly further influences the conformational behavior( arc F in Fig 2).
Discussion:
In this paper, we have investigated the influence of interfaces on the conformational behavior of two synthetic peptides, EALA and LK, along with its alanine mutant AK, with built-in secondary α-helical amphiphilicity. First, we have characterized the behavior of individual molecules in bulk water and found that they are neither random coils nor fully formed α-helices, but adopt multiple conformations and secondary structure elements with short lifetimes on the order of 100 ns or less. These peptides would—as isolated molecules in bulk water—be experimentally characterized as partially or even fully intrinsically disordered. Often it is not quite clear, to which extent intrinsically disordered systems are composed of truly random coils and to which extent they are a broad ensemble of partially folded structures with very short lifetimes. This time- and ensemble-averaging makes a clear experimental characterization extremely difficult. Our results suggest that intrinsically disordered peptides are not necessarily always fully disordered. They do populate a variety of more or less well-defined conformational states, but none of them are dominant and long-lived enough to stabilize the system into a unique fold. A change in environmental conditions typically changes the relative depths of the free-energy minima corresponding to these ensemble structures. This possibly explains why for many intrinsically disordered polypeptides certain environmental triggers induce the formation of distinct structures. The formation of β-sheet-rich amyloid fibrils upon aggregation of IDPs such as A-β or α-synuclein can be given as examples. One important class of external stimuli that induce structure formation related to secondary amphiphilicity are interfaces—both macroscopic as well as molecular ones. We first investigated the conformational behavior of LK and EALA under the influence of the macroscopic vacuum/water interface. This interface leads to a strong partitioning of hydrophobic/hydrophilic residues, which enforces a unique conformation by stabilizing the α-helical form for both model systems. In the case of the LK peptide, the bulk water ensemble is already dominated by conformations where a large fraction of the residues are in an α-helical state. On the contrary, for EALA the probability of structures with α-helical elements in the bulk water ensemble is low( but non-zero). Thus, the shift towards fully formed α-helices for both systems supports a conformational-selection & population-shift hypothesis, where the partitioning of the side chains at the interface shifts the existing equilibria and enhances the population of the α-helical structures that are( at least partially) already present. In their response to molecular interfaces presented by an aggregation partner in bulk water the peptide systems differ considerably. For LK, the molecular interface stabilizes the α-helical state, even already at a dimer level. On the other hand, EALA dimers adopt a broad distribution of transient secondary structural elements, including( a very small fraction of) α-helical elements as well as β-sheet structures. For AK the further reduced hydrophobicity of the sidechains destabilizes the dimer state entirely. Our MD simulations and PMF calculations have shown that the differences between LK, EALA and AK can be linked to the peptides’ inherent α-helical tendencies in bulk water, as well as to their different balance of hydrophobic forces, electrostatic repulsion, and intra- and intermolecular hydrogen-bonding. The simulations also allow an interesting glimpse on to the early stages of dimer formation, when the two molecules make their first contact. Not surprisingly, the first contact is established via the extended tail of one of the molecules in its disordered state as proposed in the fly-casting mechanism. This gives rise to the speculation, that a certain degree of disorder is kinetically advantageous for aggregation of these peptide systems, since it makes random encounters more likely. One might speculate that it is not entirely coincidental that so many amyloid forming peptides are intrinsically disordered in solution since( i) they are conformationally ambiguous and( ii) disorder may enhance aggregation. We find that the peptides’ aggregation behavior is also strongly affected by presence or absence of a macroscopic interface. While the LK peptides aggregate less strongly at the vacuum/water interface compared to bulk water, two EALA molecules form very stable α-helical dimers at the vacuum/water interface. The different behavior of EALA can be linked to the distribution of surface charge and hydrophobicity, and again, strong intermolecular hydrogen bonds further stabilizes the dimer. In the present work we have focused on the qualitative analysis of the interface effect on conformational behavior of model peptides. Enhanced sampling techniques could potentially be utilized to obtain a more quantitative description of the conformational space of such molecules. However, this becomes extremely challenging for processes that do have an important diffusive component such as dimer formation. In the future it would be promising to augment these atomistic simulations with additional ones on a more coarse grained( CG) level of description, giving access to biologically relevant time and length scales. However, it is essential for such CG models to correctly reflect the environment dependent conformational preference of the molecules as seen in the present work.
Small proteins and peptides have important biomedical implications with their ability to penetrate cells , disrupt cell membranes , or form—potentially neurotoxic—fibrillar aggregates . It is rather intriguing that the same peptide can take part in all these processes depending on its environment . Hence it is important to understand how the environment guides the peptide from a disordered state to an alpha-helix or beta-sheet . By studying two different peptides with computer simulations , we find that one clue to this puzzle is the state of the allegedly disordered molecule in water , which is in fact characterized by a vast number of partially folded short-lived conformations . We investigate how interaction with macroscopic interfaces affects this multi-state conformational ensemble by promoting an alpha-helix over the other conformers . Furthermore , we show that association of two peptides can also be viewed as an interaction with an interface—a molecular interface . We show that the impact of macroscopic and molecular interfaces strongly depends on their ability to enforce partitioning of hydrophobic and hydrophilic residues . The effect gets more subtle as soon as macroscopic partitioning is coupled with peptide association where two interfaces compete: the relative balance of opposing forces determines aggregate stability and structure .