NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.     

NMR and SAXS characterization of the denatured state of the chemotactic protein CheY: implications for protein folding initiation

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that approximately 25% of the residues are involved in residual structures. Conformational shifts of the alpha-protons, heteronuclear (15)N-[(1)H] NOEs and (15)N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1-70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone (1)H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme.

Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.     

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {¹H}-¹⁵N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pK_a values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/β proteins both on fast ps/ns as well as slower μs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.     

Critical role of beta-hairpin formation in protein G folding

Comparison of the folding mechanisms of proteins with similar structures but very different sequences can provide fundamental insights into the determinants of protein folding mechanisms. Despite very little sequence similarity, the approximately 60 residue IgG binding domains of protein G and protein L both consist of a single helix packed against a four-stranded sheet formed by two symmetrically disposed beta-hairpins. We demonstrate that, as in the case of protein L, one of the two beta-turns of protein G is formed and the other disrupted in the folding transition state. Unlike protein L, however, in protein G it is the second beta-turn that is formed in the folding transition state ensemble. Substitution of an Asp residue by Ala in protein G that eliminates an i,i+2 side chain-main chain hydrogen bond in the second beta-turn slows the folding rate approximately 20-fold but has virtually no effect on the unfolding rate. Taken together with previous results, these findings suggest that the presence of an intact beta-turn in the folding transition state is a consequence of the overall topology of protein L and protein G, but the particular hairpin that is formed is determined by the detailed interatomic interactions that determine the free energies of formation of the isolated beta-hairpins.     

Structure and dynamics of an acid-denatured protein G mutant

NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 degrees C, F30H-G(B1) is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding G(B1). We have assigned almost all backbone (15)N, H(N), and H(alpha) resonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both alpha-helical and beta-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for beta-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta1-beta2 hairpin of the folded state. Steady-state ?(1)H-(15)N? NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta3-beta4 hairpin, and the beta1-beta2 turn may serve as potential initiation sites for folding of G(B). Furthermore, residual structure in acid-denatured F30H-G(B1) is more extensive than in peptide fragments corresponding to the beta1-beta2, alpha-helix, and beta3-beta4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain.     

Study of the compact denatured state of a protein by molecular dynamics simulation]

The native state can be considered as a unique conformation of the protein molecule with the lowest free energy of residue contacts. In this case, all other conformations correspond to the denatured state. The degree of their compactness varies significantly. Under folding conditions, the compact denatured state rather than the random coil is in equilibrium with native protein. The balance between the main forces of protein folding, the solvophobic interactions and conformational entropy, suggests that some properties of the compact denatured state are close to those of native protein, whereas other properties are close to those of the random coil. To investigate the molecular structure of the compact denatured state, the method of molecular dynamics simulation seems to be very useful.     

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.     

A photochemically induced dynamic nuclear polarization study of denatured states of lysozyme

Photochemically induced dynamic nuclear polarization (photo-CIDNP) techniques have been used to examine denatured states of lysozyme produced under a variety of conditions. 1H CIDNP difference spectra of lysozyme denatured thermally, by the addition of 10 M urea, or by the complete reduction of its four disulfide bonds were found to differ substantially not only from the spectrum of the native protein but also from that expected for a completely unstructured polypeptide chain. Specifically, denatured lysozyme showed a much reduced enhancement of tryptophan relative to tyrosine than did a mixture of blocked amino acids with the same composition as the intact protein. By contrast, the CIDNP spectrum of lysozyme denatured in dimethyl sulfoxide solution was found to be similar to that expected for a random coil. It is proposed that nonrandom hydrophobic interactions are present within the denatured states of lysozyme in aqueous solution and that these reduce the reactivity of tryptophan residues relative to tyrosine residues. Characterization of such interactions is likely to be of considerable significance for an understanding of the process of protein folding.     

Structural characterization of a trapped folding intermediate of pyrrolidone carboxyl peptidase from a hyperthermophile

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.     

Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR

The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine ((19)F-Phe) for (19)F NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by (19)F NMR, the loss of (19)F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding (19)F NMR experiments. In double-jump (19)F NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.     

The unfolded state of the villin headpiece helical subdomain: computational studies of the role of locally stabilized structure

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36's simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first alpha-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third alpha-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second alpha-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been discussed.     

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.     

Residual structure in unfolded proteins

The denatured state ensemble (DSE) of unfolded proteins, once considered to be well-modeled by an energetically featureless random coil, is now well-known to contain flickering elements of residual structure. The position and nature of DSE residual structure may provide clues toward deciphering the protein folding code. This review focuses on recent advances in our understanding of the nature of DSE collapse under folding conditions, the quantification of the stability of residual structure in the DSE, the determination of the location and types of residues involved in thermodynamically significant residual structure and advances in detection of long-range interactions in the DSE.     

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water.Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure.The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their pK values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH₃ resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis

A simple alternative method for obtaining "random coil" chemical shifts by intrinsic referencing using the protein's own peptide sequence is presented. These intrinsic random coil backbone shifts were then used to calculate secondary chemical shifts, that provide important information on the residual secondary structure elements in the acid-denatured state of an acyl-coenzyme A binding protein. This method reveals a clear correlation between the carbon secondary chemical shifts and the amide secondary chemical shifts 3-5 residues away in the primary sequence. These findings strongly suggest transient formation of short helix-like segments, and identify unique sequence segments important for protein folding.     

Direct analysis of backbone-backbone hydrogen bond formation in protein folding transition states

Here we investigate the role of backbone-backbone hydrogen bonding interactions in stabilizing the protein folding transition states of two model protein systems, the B1 domain of protein L (ProtL) and the P22 Arc repressor. A backbone modified analogue of ProtL containing an amide-to-ester bond substitution between residues 105 and 106 was prepared by total chemical synthesis, and the thermodynamic and kinetic parameters associated with its folding reaction were evaluated. Ultimately, these parameters were used in a Phi-value analysis to determine if the native backbone-backbone hydrogen bonding interaction perturbed in this analogue (i.e. a hydrogen bond in the first beta-turn of ProtL's beta-beta-alpha-beta-beta fold) was formed in the transition state of ProtL's folding reaction. Also determined were the kinetic parameters associated with the folding reactions of two Arc repressor analogues, each containing an amide-to-ester bond substitution in the backbone of their polypeptide chains. These parameters were used together with previously established thermodynamic parameters for the folding of these analogues in Phi-value analyses to determine if the native backbone-backbone hydrogen bonding interactions perturbed in these analogues (i.e. a hydrogen bond at the end of the intersubunit beta-sheet interface and hydrogen bonds at the beginning of the second alpha-helix in Arc repressor's beta-alpha-alpha structure) were formed in the transition state of Arc repressor's folding reaction. Our results reveal that backbone-backbone hydrogen bonding interactions are formed in the beta-turn and alpha-helical transition state structures of ProtL and Arc repressor, respectively; and they were not formed in the intersubunit beta-sheet interface of Arc repressor, a region of Arc repressor's polypeptide chain previously shown to have other non-native-like conformations in Arc's protein folding transition state.