Formation Of Backbone Hydrogen Bonds Research Articles

In silico study on graphene quantum dots modified with various functional groups inhibiting α‑synuclein dimerization

Hypothesis: Graphene quantum dots (GQDs) with various functional groups are hypothesized to inhibit the α-synuclein (αS) dimerization, a crucial step in Parkinson’s disease pathogenesis. The potential of differently functionalized GQDs is systematically explored.Experiments: All-atom replica-exchange molecular dynamics simulations (accumulating to 75.6 μs) in explicit water were performed to study the dimerization of the αS non-amyloid component region and the influence of GQDs modified with various functional groups. Conformation ensemble, binding behavior, and free energy analysis were conducted.Findings: All studied GQDs inhibit β-sheet and backbone hydrogen bond formation in αS dimers, leading to looser oligomeric conformations. Charged GQDs severely impede the growth of extended β-sheets by providing extra contact surface. GQD binding primarily disrupts αS inter-peptide interactions through π-π stacking, CH-π interactions, and for charged GQDs, additionally through salt-bridge and hydrogen bonding interactions. GQD-COO– showed the most optimal inhibitory effect, binding mode, and intensity, which holds promise for the development of nanomedicines targeting amyloid aggregation in neurodegenerative diseases.

Melatonin Inhibits hIAPP Oligomerization by Preventing β-Sheet and Hydrogen Bond Formation of the Amyloidogenic Region Revealed by Replica-Exchange Molecular Dynamics Simulation.

The pathogenesis of type 2 diabetes (T2D) is highly related to the abnormal self-assembly of the human islet amyloid polypeptide (hIAPP) into amyloid aggregates. To inhibit hIAPP aggregation is considered a promising therapeutic strategy for T2D treatment. Melatonin (Mel) was reported to effectively impede the accumulation of hIAPP aggregates and dissolve preformed fibrils. However, the underlying mechanism at the atomic level remains elusive. Here, we performed replica-exchange molecular dynamics (REMD) simulations to investigate the inhibitory effect of Mel on hIAPP oligomerization by using hIAPP20–29 octamer as templates. The conformational ensemble shows that Mel molecules can significantly prevent the β-sheet and backbone hydrogen bond formation of hIAPP20–29 octamer and remodel hIAPP oligomers and transform them into less compact conformations with more disordered contents. The interaction analysis shows that the binding behavior of Mel is dominated by hydrogen bonding with a peptide backbone and strengthened by aromatic stacking and CH–π interactions with peptide sidechains. The strong hIAPP–Mel interaction disrupts the hIAPP20–29 association, which is supposed to inhibit amyloid aggregation and cytotoxicity. We also performed conventional MD simulations to investigate the influence and binding affinity of Mel on the preformed hIAPP1–37 fibrillar octamer. Mel was found to preferentially bind to the amyloidogenic region hIAPP20–29, whereas it has a slight influence on the structural stability of the preformed fibrils. Our findings illustrate a possible pathway by which Mel alleviates diabetes symptoms from the perspective of Mel inhibiting amyloid deposits. This work reveals the inhibitory mechanism of Mel against hIAPP20–29 oligomerization, which provides useful clues for the development of efficient anti-amyloid agents.

Thermodynamics of helix formation in small peptides of varying length in vacuo, in implicit solvent, and in explicit solvent.

Helix formation plays a critical role in the functionality of proteins. Various biological processes such as protein-ligand, protein-protein, and protein-nucleotide interactions are strongly influenced by the conformational stability of peptides. Small peptides represent ideal models to study the process of helix formation due to their small size and simple structures. As is widely known in the biophysical community, different amino acids have different propensities for helix formation. In this work, we used model peptides to study the peptide-length dependence and the sequence dependence of the thermodynamic profiles along the helix formation pathway in vacuo and in water. The peptides showed interesting unfolding behavior in vacuo, as this process was found to be protonation-state dependent, sequence-length dependent, and to change when residues in the peptide were mutated. Ionizable group protonation alters helix propensity by changing the degree of charge-charge repulsion in the peptide. Longer sequences tend to form more hydrogen bonds and are more likely to form helical structures. Mutation of an alanine residue to another naturally occurring amino acid changes the interactions within the peptide, which in turn alters its helix-forming tendency. By contrast, solvation leads to the generation of protein-solvent hydrogen bonds, which compensate for the energy penalty associated with the deformation of backbone hydrogen bonds in the extended conformation. Flat free-energy profiles were obtained for all mutated peptides of various lengths. Although several implicit-solvent models were able to reproduce free energies of helix formation in explicit solvent, the conformational ensembles varied with the solvent model applied, particularly in terms of the populations of secondary structures and the formation of backbone hydrogen bonds. Also, due to the absence of explicit descriptions of solvent molecules and thus protein-solvent hydrogen bonds in implicit-solvent models, they did not yield accurate hydrogen-bonding profiles.

Monitoring Backbone Hydrogen-Bond Formation in β-Barrel Membrane Protein Folding.

β-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire β-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale.

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

Abstractβ‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.

Nascent β-Hairpin Formation of a Natively Unfolded Peptide Reveals the Role of Hydrophobic Contacts

Despite the important role of the unfolded states in protein stability, folding, and aggregation, they remain poorly understood due to the lack of residue-specific experimental data. Here, we explore features of the unfolded state of the NTL9 protein by applying all-atom replica-exchange simulations to the two fragment peptides NTL9(1–22) and NTL9(6–17). We found that while NTL9(6–17) is unstructured, NTL9(1–22) transiently folds as various β-hairpins, a fraction of which contain a native β-sheet. Interestingly, despite a large number of charged residues, the formation of backbone hydrogen bonds is concomitant with hydrophobic but not electrostatic contacts. Although the fragment peptides lack a proposed specific contact between Asp8 and Lys12, the individually weak, nonspecific interactions with lysines together stabilize the charged Asp8, leading to a pKa shift of nearly 0.5 units, in agreement with the NMR data. Taken together, our data suggest that the unfolded state of NTL9 likely contains a β-hairpin in segment 1–22 with sequence-distant hydrophobic contacts, thus lending support to a long-standing hypothesis that the unfolded states of proteins exhibit native-like topology with hydrophobic clusters.

Studies of Early Events of Folding of a Predominately β-Sheet Protein Using Fluorescence Correlation Spectroscopy and Other Biophysical Methods

The interplay between the early collapse of the unfolded state and the formation of the secondary structure has been the subject of extensive research in protein chemistry. In this study, we used the intestinal fatty acid binding protein (IFABP), a small model protein with predominately β-sheet structure, to study the early events, including the early chain collapse and the formation of the secondary structure. We used a combination of fluorescence correlation spectroscopy and far-UV circular dichroism (CD) to understand how these early processes influence the late folding events like the stabilization of the secondary structure and aggregation. Acid-induced unfolded IFABP was found to collapse in the presence of low concentrations of added salt and aggregate at higher concentrations. Both the formation of the collapsed state and aggregation were conveniently probed by fluorescence correlation spectroscopy, a sensitive fluorescence technique with single-molecule resolution. In contrast, the formation of the secondary structure was monitored by far-UV CD. The results suggested that backbone hydrogen bond formation, not only the overall hydrophobicity of IFABP, may play crucial roles in the early collapse. Two mutant proteins positioned at a crucial nucleating site, namely, G80V and L64G, although being opposite in their overall hydrophobicity, collapsed relatively rapidly compared to the wild-type protein. The interconnection among the early collapse, the formation of the secondary structure, and aggregation was similar for these two mutants. Another mutant, G44V, which was identical in its overall hydrophobicity to G80V but situated in a region distant from the hydrophobic core, was found to be very different from G80V and L64G.

Ultrafast folding and molecular dynamics of a linear hydrophobic β-hairpin

The first computational study of the folding and dynamics of a hydrophobic β-hairpin containing a central heterochiral diproline segment is reported. Linear hydrophobic sequences containing centrally positioned diproline motifs, heterochiral (DL/LD) and homochiral (LL/DD)), are investigated for their ability to form β-hairpins. Heterochiral diproline motifs (LD/DL) reveal the formation of stable β-hairpins with the backbone adopting β-turn conformation and the formation of backbone hydrogen bonds with antiparallel cross-strand registry, whereas the homochiral diproline (LL/DD) containing sequences tend to adopt PPII helix conformation. The competition between the β-turn formation and the backbone H-bond ladder of the antiparallel β-strands in heterochiral diproline containing sequences is employed to validate the hypothesis that β-turn formation precedes inter-strand registry in the folding of a β-hairpin (“zipper” mechanism). The observation of noncanonical hydrogen bonds leads to a folded β-hairpin-like conformation and points to the existence of relatively stable transition state intermediates, between the unfolded (extended) and folded (β-hairpin) states. The MD simulations are in excellent agreement with the experimental studies on the model system and constitute the very first computational investigation of the folding and dynamics of a completely hydrophobic synthetic β-hairpin containing heterogeneous residues of mixed chirality.

Elucidating the Locking Mechanism of Peptides onto Growing Amyloid Fibrils through Transition Path Sampling

We investigate the molecular mechanism of monomer addition to a growing amyloid fibril composed of the main amyloidogenic region from the insulin peptide hormone, the LVEALYL heptapeptide. Applying transition path sampling in combination with reaction coordinate analysis reveals that the transition from a docked peptide to a locked, fully incorporated peptide can occur in two ways. Both routes involve the formation of backbone hydrogen bonds between the three central amino acids of the attaching peptide and the fibril, as well as a reorientation of the central Glu side chain of the locking peptide toward the interface between two β-sheets forming the fibril. The mechanisms differ in the sequence of events. We also conclude that proper docking is important for correct alignment of the peptide with the fibril, as alternative pathways result in misfolding.

Accessible Surfaces of Beta Proteins Increase with Increasing Protein Molecular Mass More Rapidly than Those of Other Proteins

Here we present a systematic analysis of accessible surface areas and hydrogen bonds of 2554 globular proteins from four structural classes (all-α, all-β, α/β and α+β proteins) that is aimed to learn in which structural class the accessible surface area increases with increasing protein molecular mass more rapidly than in other classes, and what structural peculiarities are responsible for this effect. The beta structural class of proteins was found to be the leader, with the following possible explanations of this fact. First, in beta structural proteins, the fraction of residues not included in the regular secondary structure is the largest, and second, the accessible surface area of packaged elements of the beta-structure increases more rapidly with increasing molecular mass in comparison with the alpha-structure. Moreover, in the beta structure, the probability of formation of backbone hydrogen bonds is higher than that in the alpha helix for all residues of α+β proteins (the average probability is 0.73±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without proline) and α/β proteins, except for asparagine, aspartic acid, glycine, threonine, and serine (0.70±0.01 for the beta-structure and 0.60±0.01 for the alpha-structure without the proline residue). There is a linear relationship between the number of hydrogen bonds and the number of amino acid residues in the protein ().

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.

Temperature Dependence of Hydrogen-Bond Stability in β-Hairpin Structures

Understanding the temperature effect in the folding of multiple β-hairpins with different sequence (although based on an approximate solution model) makes possible quantitative characterization of the different contributing factors that are difficult to be obtained from single temperature studies. The detailed thermodynamics analyses performed in this article provide at least a semiquantitative understanding of how temperature and positions affect the stability of individual backbone hydrogen bonds in β-hairpin structures. These effects are then rationalized, according to the separation into enthalpic and entropic contributions. The formation of backbone hydrogen bonds at the terminal position is favored at low temperatures and those near the turn become more favorable at high temperatures, as a result of the differences in their formation entropy. Regardless of the differences in the turn stability, the side-chain hydrophobicity, and the room temperature folding mechanism of these β-hairpins, there is a shift to the “zip-out” mechanism in the assembling of backbone hydrogen bonds as temperature increases for all polypeptides under study. In addition, it was also observed that although the backbone hydrogen-bond formation shows a strong dependence on temperature, the formation order of the three structural elements of β-hairpin (the turn, hydrophobic core cluster, and hydrogen-bond assembly) along the minimum free energy pathway in the free energy landscapes appears to be only sequence dependent and largely unaffected by the temperature change.

Protein β-Sheet Nucleation Is Driven by Local Modular Formation

Despite its central role in the protein folding process, the specific mechanism(s) behind beta-sheet formation has yet to be determined. For example, whether the nucleation of beta-sheets, often containing strands separated in sequence by many residues, is local or not remains hotly debated. Here, we investigate the initial nucleation step of beta-sheet formation by performing an analysis of the smallest beta-sheets in a non-redundant dataset on the grounds that the smallest sheets, having undergone little growth after nucleation, will be enriched for nucleating characteristics. We find that the residue propensities are similar for small and large beta-sheets as are their interstrand pairing preferences, suggesting that nucleation is not primarily driven by specific residues or interacting pairs. Instead, an examination of the structural environments of the two-stranded sheets shows that virtually all of them are contained in single, compact structural modules, or when multiple modules are present, one or both of the chain termini are involved. We, therefore, find that beta-nucleation is a local phenomenon resulting either from sequential or topological proximity. We propose that beta-nucleation is a result of two opposite factors; that is, the relative rigidity of an associated folding module that holds two stretches of coil close together in topology coupled with sufficient chain flexibility that enables the stretches of coil to bring their backbones in close proximity. Our findings lend support to the hydrophobic zipper model of protein folding (Dill, K. A., Fiebig, K. M., and Chan, H. S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1942-1946). Implications for protein folding are discussed.

Importance of Protein Context on the Unfolding Pathways of β-hairpins

Small β-hairpin peptides as well as three-stranded WW domains have been used as models for the folding of β-hairpins in larger proteins. Previous studies of the FBP28 WW domain proposed that side chain contacts between residues in the strands and not the precise order of backbone hydrogen bond formation guide β-hairpin folding. But how applicable is the folding of model systems, such as FBP28 WW domain, to the folding of β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding molecular dynamics simulations of three proteins that share a double hairpin motif structurally similar to WW domains: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain.

Induced β-Barrel Formation of the Alzheimer's A β25–35 Oligomers on Carbon Nanotube Surfaces: Implication for Amyloid Fibril Inhibition

Recent experimental studies show that carbon nanotubes impact the aggregation process of proteins associated with neurodegenerative diseases. However, the details of molecular interactions between proteins and carbon nanotubes are still not well understood. In this study, we investigate the initial adsorption features and dynamics of the Alzheimer's amyloid- β peptide spanning residues 25–35 (A β25–35) on a single-walled carbon nanotube (SWNT) surface using fully atomic molecular dynamics simulations (MD) in explicit solvent. The initial configurations of the A β25–35 peptides consist of two preformed bilayer β-sheets, each with four or five β-strands in parallel or mixed antiparallel-parallel orientations. Our simulations show, for what we believe is the first time, that two disjointed A β25–35 β-sheets with mixed antiparallel-parallel strands can assemble into β-barrels wrapping the SWNT. In contrast, both simulations of A β25–35 without SWNT, and simulations of SWNT−A β25–35 with purely parallel β-strands, lead to disordered aggregates. We find that A β25–35 β-barrel formation involves at least two steps: i), curving of the A β25–35 β-sheets as a result of strong hydrophobic interactions with carbon nanotube concomitantly with dehydration of the SWNT-peptide interface; and ii), intersheet backbone hydrogen bond formation with fluctuating intrasheet hydrogen bonds. Detailed analysis of the conversion shows that β-barrel formation on SWNT surface results from the interplay of dehydration and peptide-SWNT/peptide-peptide interactions. Implications of our results on amyloid fibril inhibition are discussed.

Probing the Folding Transition State Structure of the Villin Headpiece Subdomain via Side Chain and Backbone Mutagenesis

Backbone-backbone hydrogen bonds are a common feature of native protein structures, yet their thermodynamic and kinetic influence on folding has long been debated. This is reflected by the disparity between current protein folding models, which place hydrogen bond formation at different stages along the folding trajectory. For example, previous studies have suggested that the denatured state of the villin headpiece subdomain contains a residual helical structure that may provide a bias toward the folded state by confining the conformational search associated with its folding. Although helical hydrogen bonds clearly stabilize the folded state, here we show, using an amide-to-ester mutation strategy, that the formation of backbone hydrogen bonds within helices is not rate-limiting in the folding of the subdomain, thereby suggesting that such hydrogen bonds are unlikely to be formed en route from the denatured to the transition state. On the other hand, elimination of hydrogen bonds within the turn region elicits a slower folding rate, consistent with the hypothesis that these residues are involved in the formation of a folding nucleus. While illustrating a potentially conserved aspect of helix-turn-helix folding, our results further underscore the inherent importance of turns in protein supersecondary structure formation.

Single Molecule Force Spectroscopy and Steered Molecular Dynamics Simulations Reveal the Mechanical Design of the Third FnIII Domain of Tenascin-C

By combining single molecule atomic force microscopy, proline mutagenesis and steered molecular dynamics simulations, we investigate the mechanical unfolding dynamics and mechanical design of the third FnIII domain of tenascin-C (TNfn3) in detail. The mechanical stability of TNfn3 is found to be similar to that of other constituting FnIII domains of tenascin-C, and the unfolding process of TNfn3 is an apparent two-state process. The hydrophobic core packing of TNfn3 was previously reported as the key element of its mechanical stability. Here, employing proline mutagenesis to block the formation of backbone hydrogen bonds and introduce structural disruption in β sheet, we showed that not only hydrophobic core packing plays important roles in determining the mechanical stability of TNfn3, backbone hydrogen bonds in β hairpins are also responsible for the overall mechanical stability of TNfn3. Furthermore, proline mutagenesis revealed that the mechanical design of TNfn3 is very robust and proline substitution in β sheets only leads to mild reduction in mechanical stability. We also compare the AFM results with those of SMD simulations to understand the molecular details underlying the mechanical unfolding of TNfn3. We found that the mechanical unfolding and design of TNfn3 is significantly different from its structural homologue the tenth FnIII domain from fibronectin. These results serve as a starting point for systematically analyzing the mechanical architecture of other FnIII domains in tenascins-C and will help to gain a better understanding of some of the complex features observed for the stretching of native tenascin-C.

Mechanical Design of the Third FnIII Domain of Tenascin-C

By combining single-molecule atomic force microscopy (AFM), proline mutagenesis and steered molecular dynamics (SMD) simulations, we investigated the mechanical unfolding dynamics and mechanical design of the third fibronectin type III domain of tenascin-C (TNfn3) in detail. We found that the mechanical stability of TNfn3 is similar to that of other constituting FnIII domains of tenascin-C, and the unfolding process of TNfn3 is an apparent two-state process. By employing proline mutagenesis to block the formation of backbone hydrogen bonds and introduce structural disruption in β sheet, we revealed that in addition to the important roles played by hydrophobic core packing, backbone hydrogen bonds in β hairpins are also responsible for the overall mechanical stability of TNfn3. Furthermore, proline mutagenesis revealed that the mechanical design of TNfn3 is robust and the mechanical stability of TNfn3 is very resistant to structural disruptions caused by proline substitutions in β sheets. Proline mutant F88P is one exception, as the proline mutation at position 88 reduced the mechanical stability of TNfn3 significantly and led to unfolding forces of < 20 pN. This result suggests that Phe88 is a weak point of the mechanical resistance for TNfn3. We used SMD simulations to understand the molecular details underlying the mechanical unfolding of TNfn3. The comparison between the AFM results and SMD simulations revealed similarities and discrepancies between the two. We compared the mechanical unfolding and design of TNfn3 and its structural homologue, the tenth FnIII domain from fibronectin. These results revealed the complexity underlying the mechanical design of FnIII domains and will serve as a starting point for systematically analyzing the mechanical architecture of other FnIII domains in tenascins-C, and will help to gain a better understanding of some of the complex features observed for the stretching of native tenascin-C.

Side-Chain and Backbone Ordering in Homopolymers

In order to study the relation between backbone and side-chain ordering in proteins, we have performed multicanonical simulations of deka-peptide chains with various side groups. Glu(10), Gln(10), Asp(10), Asn(10), and Lys(10) were selected to cover a wide variety of possible interactions between the side chains of the monomers. All homopolymers undergo helix-coil transitions. We found that peptides with long side chains that are capable of hydrogen bonding, i.e., Glu(10), and Gln(10), exhibit a second transition at lower temperatures connected with side-chain ordering. This occurs in the gas phase as well as in solvent, although the character of the side-chain structure is different in each case. However, in polymers with short side chains capable of hydrogen bonding, i.e., Asp(10) and Asn(10), side-chain ordering takes place over a wide temperature range and exhibits no phase transition-like character. Moreover, non-backbone hydrogen bonds show enhanced formation and fluctuations already at the helix-coil transition temperature, indicating competition between side-chain and backbone hydrogen bond formation. Again, these results are qualitatively independent of the environment. Side-chain ordering in Lys(10), whose side groups are long and polar, also takes place over a wide temperature range and exhibits no phase transition-like character in both environments. Reasons for the observed chain length threshold and consequences from these results for protein folding are discussed.

Kinetic Isotope Effects Reveal the Presence of Significant Secondary Structure in the Transition State for the Folding of the N-terminal Domain of L9

Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (ϕ value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small α−β protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its T m and Δ G 0 of unfolding. The folding rate also decreased. The parameter Φ H/D, defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D 2O background and 0.75 in a H 2O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the β-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.