Intermolecular β-Strand Networks Avoid Hub Residues and Favor Low Interconnectedness: A Potential Protection Mechanism against Chain Dissociation upon Mutation

Altogether few protein oligomers undergo a conformational transition to a state that impairs their function and leads to diseases. But when it happens, the consequences are not harmless and the so-called conformational diseases pose serious public health problems. Notorious examples are the Alzheimer''s disease and some cancers associated with a conformational change of the amyloid precursor protein (APP) and of the p53 tumor suppressor, respectively. The transition is linked with the propensity of β-strands to aggregate into amyloid fibers. Nevertheless, a huge number of protein oligomers associate chains via β-strand interactions (intermolecular β-strand interface) without ever evolving into fibers. We analyzed the layout of 1048 intermolecular β-strand interfaces looking for features that could provide the β-strands resistance to conformational transitions. The interfaces were reconstructed as networks with the residues as the nodes and the interactions between residues as the links. The networks followed an exponential decay degree distribution, implying an absence of hubs and nodes with few links. Such layout provides robustness to changes. Few links per nodes do not restrict the choices of amino acids capable of making an interface and maintain high sequence plasticity. Few links reduce the “bonding” cost of making an interface. Finally, few links moderate the vulnerability to amino acid mutation because it entails limited communication between the nodes. This confines the effects of a mutation to few residues instead of propagating them to many residues via hubs. We propose that intermolecular β-strand interfaces are organized in networks that tolerate amino acid mutation to avoid chain dissociation, the first step towards fiber formation. This is tested by looking at the intermolecular β-strand network of the p53 tetramer.     

Antiparallel Triple-strand Architecture for Prefibrillar Aβ42 Oligomers

Aβ42 oligomers play key roles in the pathogenesis of Alzheimer disease, but their structures remain elusive partly due to their transient nature. Here, we show that Aβ42 in a fusion construct can be trapped in a stable oligomer state, which recapitulates characteristics of prefibrillar Aβ42 oligomers and enables us to establish their detailed structures. Site-directed spin labeling and electron paramagnetic resonance studies provide structural restraints in terms of side chain mobility and intermolecular distances at all 42 residue positions. Using these restraints and other biophysical data, we present a novel atomic-level oligomer model. In our model, each Aβ42 protein forms a single β-sheet with three β-strands in an antiparallel arrangement. Each β-sheet consists of four Aβ42 molecules in a head-to-tail arrangement. Four β-sheets are packed together in a face-to-back fashion. The stacking of identical segments between different β-sheets within an oligomer suggests that prefibrillar oligomers may interconvert with fibrils via strand rotation, wherein β-strands undergo an ∼90° rotation along the strand direction. This work provides insights into rational design of therapeutics targeting the process of interconversion between toxic oligomers and non-toxic fibrils.     

Intrinsic α‐helical and β‐sheet conformational preferences: A computational case study of alanine

A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle (-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (N–C_α–C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the “bridge” region of-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between -ψ conformations and τ.     

Alzheimer Aβ peptide interactions with lipid membranes: Fibrils,oligomers and polymorphic amyloid channels

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Solid-state NMR reveals a close structural relationship between amyloid-β protofibrils and oligomers

We have studied tertiary contacts in protofibrils and mature fibrils of amyloid-β (Aβ) peptides using solid-state NMR spectroscopy. Although intraresidue contacts between Glu-22 and Ile-31 were found in Aβ protofibrils, these contacts were completely absent in mature Aβ fibrils. This is consistent with the current models of mature Aβ fibrils. As these intramolecular contacts have also been reported in Aβ oligomers, our measurements suggest that Aβ protofibrils are structurally more closely related to oligomers than to mature fibrils. This suggests that some structural alterations have to take place on the pathway from Aβ oligomers/protofibrils to mature fibrils, in agreement with a model that suggests a conversion of intramolecular hydrogen-bonded structures of Aβ oligomers to the intermolecular stabilized mature fibrils (Hoyer, W., Grönwall, C., Jonsson, A., Ståhl, S., and Härd, T. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5099–5104).     

Evolution rescues folding of human immunodeficiency virus-1 envelope glycoprotein GP120 lacking a conserved disulfide bond

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues.     

A 3D model of the voltage-dependent anion channel (VDAC)

Eukaryotic porins are a group of membrane proteins whose best known role is to form an aqueous pore channel in the mitochondrial outer membrane. As opposed to the bacterial porins (a large family of protein whose 3D structure has been determined by X-ray diffraction), the structure of eukaryotic porins (also termed VDACs, voltage-dependent anion-selective channels) is still a matter of debate. We analysed the secondary structure of VDAC from the yeast Saccharomyces cerevisiae, the fungus Neurospora crassa and the mouse with different types of neural network-based predictors. The predictors were able to discriminate membrane β-strands, globular -helices and membrane -helices and localised, in all three VDAC sequences, 16 β-strands along the chain. For all three sequences the N-terminal region showed a high propensity to form a globular -helix. The 16 β-strand VDAC motif was thus aligned to a bacterial porin-derived template containing a similar 16 β-strand motif. The alignment of the VDAC sequence with the bacterial porin sequence was used to compute a set of 3D coordinates, which constitutes the first 3D prediction of a eukaryotic porin. All the predicted structures assume a β-barrel structure composed of 16 β-strands with the N-terminus outside the membrane. Loops are shorter in this side of the membrane than in the other, where two long loops are protruding. The shape of the pore varies between almost circular for Neurospora and mouse and slightly oval for yeast. Average values between 3 and 2.5 nm at the C-carbon backbone are found for the diameter of the channels. In this model VDAC shows large portions of the structure exposed on both sides of the membrane. The architecture we determine allows speculation about the mechanism of possible interactions between VDAC and other proteins on both sides of the mitochondrial outer membrane. The computed 3D model is consistent with most of the experimental results so far reported.     

Lone pair ··· π interactions between water oxygens and aromatic residues: Quantum chemical studies based on high‐resolution protein structures and model compounds

The π electron cloud of aromatic centers is known to be involved in several noncovalent interactions such as C—H···π, O—H···π, and π···π interactions in biomolecules. Lone-pair (lp) ··· π interactions have gained attention recently and their role in biomolecular structures is being recognized. In this article, we have carried out systematic analysis of high-resolution protein structures and identified more than 400 examples in which water oxygen atoms are in close contact (distance < 3.5 Å) with the aromatic centers of aromatic residues. Three different methods were used to build hydrogen atoms and we used a consensus approach to find out potential candidates for lp···π interactions between water oxygen and aromatic residues. Quantum mechanical calculations at MP2/6-311++G(d,p) level on model systems based on protein structures indicate that majority of the identified examples have energetically favorable interactions. The influence of water hydrogen atoms was investigated by sampling water orientations as a function of two parameters: distance from the aromatic center and the angle between the aromatic plane and the plane formed by the three water atoms. Intermolecular potential surfaces were constructed using six model compounds representing the four aromatic amino acids and 510 different water orientations for each model compound. Ab initio molecular orbital calculations at MP2/6-311++G(d,p) level show that the interaction energy is favorable even when hydrogen atoms are farthest from the aromatic plane while water oxygen is pointing toward the aromatic center. The strength of such interaction depends upon the distance of water hydrogen atoms from the aromatic substituents. Our calculations clearly show that the lp···π interactions due to the close approach of water oxygen and aromatic center are influenced by the positions of water hydrogen atoms and the aromatic substituents.     

Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein

Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrP^Sc) converted from a normal host cellular prion protein (PrP^C). Experimental studies suggest that PrP^C is enriched with α-helical structure, whereas PrP^Sc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrP^C leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface.     

Mechanisms of hybrid oligomer formation in the pathogenesis of combined Alzheimer's and Parkinson's diseases

Background

Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer''s disease (AD) and Parkinson''s disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear.

Methodology/Principal Findings

In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.

Conclusion/Significance

These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.     

Crystal structure of the yeast metacaspase yca1

Yca1, the only metacaspase in Saccharomyces cerevisiae, is thought to be a clan CD cysteine protease that includes the caspase subfamily. Although yeast is a single cell eukaryote, it can undergo a cell death process reminiscent of apoptosis. Yca1 has been reported to play an important role in the regulation of such apoptotic process. However, the structure and functional mechanism of Yca1 remain largely enigmatic. In this study, we report the crystal structure of the Yca1 metacaspase at 1.7 Å resolution, confirming a caspase-like fold. In sharp contrast to canonical caspases, however, Yca1 exists as a monomer both in solution and in the crystals. Canonical caspase contains six β-strands, with strand β6 pairing up with β6 of another caspase molecule to form a homodimerization interface. In Yca1, an extra pair of antiparallel β-strands forms a continuous β-sheet with the six caspase-common β-strands, blocking potential dimerization. Yca1 was reported to undergo autocatalytic processing in yeast; overexpression in bacteria also led to autoprocessing of Yca1 into two fragments. Unexpectedly, we found that both the autocatalytic processing and the proteolytic activity of Yca1 are greatly facilitated by the presence of calcium (Ca²⁺), but not other divalent cations. Our structural and biochemical characterization identifies Yca1 as a Ca²⁺-activated cysteine protease that may cleave specific substrates during stress response in yeast.     

Patterns in ionizable side chain interactions in protein structures

In a selected set of 44 high-resolution, non-homologous protein structures, the intramolecular hydrogen bonds or salt bridges formed by ionizable amino acid side chains were identified and analyzed. The analysis was based on the investigation of several properties of the involved residues such as their solvent exposure, their belonging to a certain secondary structural element, and their position relative to the N- and C-termini of their respective structural element. It was observed that two-thirds of the interactions made by basic or acidic side chains are hydrogen bonds to polar uncharged groups. In particular, the majority (78%) of the hydrogen bonds between ionizable side chains and main chain polar groups (sch:mch bonds) involved at least one buried atom, and in 42% of the cases both interacting atoms were buried. In α-helices, the sch:mch bonds observed in the proximity of the C- and N-termini show a clear preference for acidic and basic side chains, respectively. This appears to be due to the partial charges of peptide group atoms at the termini of α-helices, which establish energetically favorable electrostatic interactions with side chain carrying opposite charge, at distances even greater than 4.5 Å. The sch:mch interactions involving ionizable side chains that belong either to β-strands or to the central part of α-helices are based almost exclusively on basic residues. This results from the presence of main chain carbonyl oxygen atoms in the protein core which have unsatisfied hydrogen bonding capabilities.     

A Foldamer-Dendrimer Conjugate Neutralizes Synaptotoxic β-Amyloid Oligomers

Background and Aims

Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimer''s disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge.

Methods and Results

Short helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration.

Conclusions

The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for?the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices α5 and α6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.     

On the occurrence of linear groups in proteins

Linear groups—polypeptide conformations based on a single repeating φ,ψ-pair—are a foundational concept in protein structure, yet how they are presented in textbooks is based largely on theoretical studies from the early days of protein structure analysis. Now, ultra-high resolution protein structures provide a resource for an accurate empirical and systematic assessment of the linear groups that truly exist in proteins. Here, a purely conformation-based survey of linear groups shows that only three distinct φ,ψ-regions occur: a diverse set of extended conformations mostly present as β-strands, a broad population of polyproline-II-like spirals, and a tight cluster that includes the highly populated α-helix and the conformationally-similar but much less populated 3₁₀-helix. Rare, short left-handed α-/3₁₀-helical turns with repeating φ,ψ-angles occur, but none are longer than three residues. Misperceptions dispelled by this study are the existence of 2.2₇- and π-helices as linear groups, the existence of specific ideal φ,ψ-angles for each linear group, and the existence of a substantive difference in the φ,ψ-preferences for parallel versus antiparallel β-strands. This study provides a concrete basis for updating and enhancing how we think about and teach the basics of protein structure.     

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of β-pore-forming toxins such as aerolysin, C. perfringens ϵ-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of β-strands, two of which span its entire length. Domain II and domain III have three short β-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the β-strands. The sequence of amino acids composing the α-helix and preceding β-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming β-barrel-made pores. These structural features imply that CPE is a β-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long β-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.     

Structural Insights into Amyloid Oligomers of the Parkinson Disease-related Protein α-Synuclein

The presence of intraneuronal deposits mainly formed by amyloid fibrils of the presynaptic protein α-synuclein (AS) is a hallmark of Parkinson disease. Currently, neurotoxicity is attributed to prefibrillar oligomeric species rather than the insoluble aggregates, although their mechanisms of toxicity remain elusive. Structural details of the supramolecular organization of AS oligomers are critically needed to decipher the structure-toxicity relationship underlying their pathogenicity. In this study, we employed site-specific fluorescence to get a deeper insight into the internal architecture of AS oligomeric intermediates. We demonstrate that AS oligomers are ordered assemblies possessing a well defined pattern of intermolecular contacts. Some of these contacts involve regions that form the β-sheet core in the fibrillar state, although their spatial arrangement may differ in the two aggregated forms. However, even though the two termini are excluded from the fibrillar core, they are engaged in a number of intermolecular interactions within the oligomer. Therefore, substantial structural remodeling of early oligomeric interactions is essential for fibril growth. The intermolecular contacts identified in AS oligomers can serve as targets for the rational design of anti-amyloid compounds directed at preventing oligomeric interactions/reorganizations.     

Membrane channel forming polypeptides. 270-MHz proton magnetic resonance studies of the aggregation of the 11-21 fragment of suzukacillin in organic solvents

270-MHz 1H NMR studies of the 11-21 suzukacillin fragment Boc-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (11-G) and its analogue Boc-Ala-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (11-A) have been carried out in CDCl3 and (CD3)2SO. The NH chemical shifts and their temperature coefficients have been measured as a function of peptide concentration in both solvents. It is established that replacement of Gln by Ala is without effect on backbone conformation. Both peptides adopt highly folded 310 helical conformations stabilized by seven intramolecular 4 leads to hydrogen bonds. Nonlinear temperature dependences are demonstrated for free NH groups in the Gln(1) peptide. Aggregation is mediated by intermolecular hydrogen bonds formed by solvent-exposed NH groups. A major role for the Gln side chain in peptide association is suggested by differences in the NMR behavior of the Gln(1) and Ala(1) peptides. For the Gln(1) peptide in CDCl3, the carboxamide side chain carbonyl group forms an intramolecular hydrogen bond to the peptide backbone, while the trans side chain NH shows evidence for intermolecular interactions. In (CD3)2SO, the cis carboxamide NH is involved in intermolecular hydrogen bonding. The possible role of the central Gln residue in stabilizing aggregates of peptide channel formers is discussed, and a model for hexameric association is postulated.