Conversion of type I 4:6 to 3:5 beta-turn types in human acidic fibroblast growth factor: effects upon structure, stability, folding, and mitogenic function

Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 contains 11 beta-turns, the majority being type I 3:5; however, a type I 4:6 turn is also found at three symmetry-related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of deletion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional sequence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local environment). Although the results suggest that a stable, foldable beta-trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn 1 of FGF-1 appears essential for efficient mitogenic function.     

Role of amino acid residues at turns in the conformational stability and folding of human lysozyme

To clarify the role of amino acid residues at turns in the conformational stability and folding of a globular protein, six mutant human lysozymes deleted or substituted at turn structures were investigated by calorimetry, GuHCl denaturation experiments, and X-ray crystal analysis. The thermodynamic properties of the mutant and wild-type human lysozymes were compared and discussed on the basis of their three-dimensional structures. For the deletion mutants, Delta47-48 and Delta101, the deleted residues are in turns on the surface and are absent in human alpha-lactalbumin, which is homologous to human lysozyme in amino acid sequence and tertiary structure. The stability of both mutants would be expected to increase due to a decrease in conformational entropy in the denatured state; however, both proteins were destabilized. The destabilizations were mainly caused by the disappearance of intramolecular hydrogen bonds. Each part deleted was recovered by the turn region like the alpha-lactalbumin structure, but there were differences in the main-chain conformation of the turn between each deletion mutant and alpha-lactalbumin even if the loop length was the same. For the point mutants, R50G, Q58G, H78G, and G37Q, the main-chain conformations of these substitution residues located in turns adopt a left-handed helical region in the wild-type structure. It is thought that the left-handed non-Gly residue has unfavorable conformational energy compared to the left-handed Gly residue. Q58G was stabilized, but the others had little effect on the stability. The structural analysis revealed that the turns could rearrange the main-chain conformation to accommodate the left-handed non-Gly residues. The present results indicate that turn structures are able to change their main-chain conformations, depending upon the side-chain features of amino acid residues on the turns. Furthermore, stopped-flow GuHCl denaturation experiments on the six mutants were performed. The effects of mutations on unfolding-refolding kinetics were significantly different among the mutant proteins. The deletion/substitutions in turns located in the alpha-domain of human lysozyme affected the refolding rate, indicating the contribution of turn structures to the folding of a globular protein.     

Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor

Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.     

Accommodation of a highly symmetric core within a symmetric protein superfold

An alternative core packing group, involving a set of five positions, has been introduced into human acidic FGF-1. This alternative group was designed so as to constrain the primary structure within the core region to the same threefold symmetry present in the tertiary structure of the protein fold (the beta-trefoil superfold). The alternative core is essentially indistinguishable from the WT core with regard to structure, stability, and folding kinetics. The results show that the beta-trefoil superfold is compatible with a threefold symmetric constraint on the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new core arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of beta-trefoil proteins by using symmetric structural complementarity. Remaining asymmetry within the core appears to be related to asymmetry in the tertiary structure associated with receptor and heparin binding functionality of the growth factor.     

Structural analysis of fertilin(beta) cyclic peptide mimics that are ligands for alpha6beta1 integrin.

The NMR structural analysis of two fertilin(beta) mimics cyclo(EC2DC1)YNH2, 1, and cyclo(D2EC2D1C1)YNH2, 2 is described. Both of these mimics are moderate inhibitors of sperm-egg binding with IC50 values of 500 microm in a mouse in vitro fertilization assay. For peptide 1, the optimized conformations that best match the NMR data have a pseudo-type II' beta-turn with the linker and Glu at the i+1 and i+2 positions, respectively. The EC2D1C1 sequence is in a nonclassical (type IV) beta-turn. For peptide 2, the conformation that best matches the NMR data has two turns: a pseudo-type II' beta-turn in the D2EC2D1 sequence followed by a nonclassical beta-turn in the EC2D1C1 sequence. The Cbeta-Cbeta distance between E and D1 in peptide 1 is 9.1 A, in peptide 2, it is 7.7 A. Thus, one possibility for the high IC50 values of these cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn, and thus much entropy must still be lost upon binding to the alpha6beta1 integrin. This explains why the cyclic peptides are the same as linear peptides at inhibiting sperm-egg binding.     

Folding of alpha(r)beta and epsilonbeta reverse turns; a nanosecond molecular dynamics simulation of the hexapeptide MSALNT and the octapeptide NMSALNTL in water.

Folding of the hexapeptide MSALNT and the octapeptide NMSALNTL were investigated using 2.8 ns molecular dynamics (MD) simulations in aqueous solution. In the simulation, the central sequence SALN of the hexapeptide folded rapidly within 200 ps into an alpha(r)beta turn conformation (type VIII conformation) and remained in this conformation for the rest of the trajectory. The sequence SALN of the octapeptide needed 2 ns to fold via epsilonbeta conformations into a similar conformation. The results join the sequences into a growing group of sequences which have a tendency to form secondary structures and thereby to direct protein folding. The structures of the reverse turn conformations were in accordance with the experimental results (Hakalehto et al., Eur J. Biochem. 250, 19-29 (1997)). The main driving force of folding seems to be the hydrophobic interaction between the side chains of Ala and Leu at the i+1 and i+2 positions of the beta-turn.     

Accurate computer-based design of a new backbone conformation in the second turn of protein L.

The rational design of loops and turns is a key step towards creating proteins with new functions. We used a computational design procedure to create new backbone conformations in the second turn of protein L. The Protein Data Bank was searched for alternative turn conformations, and sequences optimal for these turns in the context of protein L were identified using a Monte Carlo search procedure and an energy function that favors close packing. Two variants containing 12 and 14 mutations were found to be as stable as wild-type protein L. The crystal structure of one of the variants has been solved at a resolution of 1.9 A, and the backbone conformation in the second turn is remarkably close to that of the in silico model (1.1 A RMSD) while it differs significantly from that of wild-type protein L (the turn residues are displaced by an average of 7.2 A). The folding rates of the redesigned proteins are greater than that of the wild-type protein and in contrast to wild-type protein L the second beta-turn appears to be formed at the rate limiting step in folding.     

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Folding of a beta-hairpin peptide derived from the N-terminus of ubiquitin. Conformational preferences of beta-turn residues dictate non-native beta-strand interactions.

The role of the non-native beta-turn sequence (NPDG) in nucleating the folding of a beta-hairpin peptide derived from the N-terminus of ubiquitin, has been examined by NMR and CD spectroscopy. The NPDG sequence, while representing a common two-residue type I turn sequence in proteins, folds to give a G1-bulged type I turn in the context of a beta-hairpin peptide, to the exclusion of other possible conformations. The turn conformation results in misalignment of the two beta strands and a beta hairpin with non-native side chain interactions. A truncated 12-residue analogue of the hairpin, in which the majority of residues in the N-terminal beta strand have been deleted, shows some weak propensity to fold into a G-bulged type I turn conformation in the absence of interstrand stabilizing interactions. The NPDG turn sequence pays some of the entropic cost in initiating folding allowing interstrand interactions, which in this case arise from the non-native pairing of residue side chains, to stabilize a significant population of the folded state. Examination of the relative abundance of the Pro-Asp type I turn, with G in the +B1 position, vs. the type I G-bulged turn PXG, in a database of high resolution structures, reveals 48 instances of PXG bulged turns for which X = Asp is by far the most common residue with 20 occurrences. Strikingly, there are no examples of a type I PD turn with G at the +B1 position, in good agreement with our experimental observations that the PDG G-bulged turn is populated preferentially in solution.     

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

In the N2 domain of the gene-3-protein of phage fd, two consecutive β-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I′ turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol^− 1 and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I′ turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I′ turn and that the other turns maintained their type I′ conformation after sequence optimization.     

Single-site mutations induce 3D domain swapping in the B1 domain of protein L from Peptostreptococcus magnus.

BACKGROUND: Thermodynamic and kinetic studies of the Protein L B1 domain (Ppl) suggest a folding pathway in which, during the folding transition, the first beta hairpin is formed while the second beta hairpin and the alpha helix are largely unstructured. The same mutations in the two beta turns have opposite effects on the folding and unfolding rates. Three of the four residues composing the second beta turn in Ppl have consecutive positive phi angles, indicating strain in the second beta turn. RESULTS: We have determined the crystal structures of the beta turn mutants G55A, K54G, and G15A, as well as a core mutant, V49A, in order to investigate how backbone strain affects the overall structure of Ppl. Perturbation of the hydrophobic interactions at the closed interface by the V49A mutation triggered the domain swapping of the C-terminal beta strand that relieved the strain in the second beta turn. Interestingly, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escalated the strain in the second beta turn, and this increased strain shifted the equilibrium toward the domain-swapped dimer. The K54G structure revealed that the increased stability is due to the reduction of strain in the second beta turn, while the G15A structure showed that increased strain alone is insufficient to trigger domain swapping. CONCLUSIONS: Domain swapping in Ppl is determined by the balance of two opposing components of the free energy. One is the strain in the second beta turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A single-site mutation can disrupt this balance and trigger domain swapping.     

Circular dichroism of beta turns in peptides and proteins.

Circular dichroism (CD) spectra are reported for two groups of cyclic hexapeptides having beta turns whose geometry can be firmly established by X-ray crystallography and by NMR spectroscopy. One series contains the sequence L-Pro-D-Phe in the geometry of the classical type II beta turn, while the second group has the sequence D-Phe-L-Pro in the closely related geometry of the gramicidin S turn. CD data on the hydrogenated peptides show that in neither series do Cotton effects due to the aromatic phenylalanyl chromophore make a significant contribution to the spectra in the 195--240-nm region. In spite of the close geometric similarity of the beta turns of these two groups of peptides, their CD spectra are quite distinct. Furthermore, comparison of our data with the CD spectra of published models for beta-turn structures suggests that it may not be possible to characterize the contribution of all beta turns to the CD spectra of proteins by a single model curve. the CD spectra of model beta turns will be more useful in characterizing the folding of oligopeptides and sequence polypeptides, where a single type of turn is present.     

Aggregation and secondary structure of synthetic amyloid beta A4 peptides of Alzheimer's disease

The deposition of amyloid beta A4 in the brain is a major pathological hallmark of Alzheimer's disease. Amyloid beta A4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates. Using synthetic peptides corresponding to the natural beta A4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of beta A4. A comparison of amino-terminally truncated beta A4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid beta A4. Infrared spectroscopy of beta A4 peptides in the solid state shows that their secondary structure consists of a beta-turn flanked by two strands of antiparallel beta-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative beta-turn positions. Limited proteolysis of these analogs allowed a localization of the central beta-turn at residues 26 to 29 of the entire sequence. Purified beta A4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a beta-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a beta-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the beta A4 sequence. This model is sustained by the properties of beta A4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between beta A4 peptides.     

Stability and amino acid preferences of type VIII reverse turn: the most common turn in peptides?

Free energies of the alpha(r)beta and betabeta conformations of 14 tetrapeptides, based on the sequence SALN and protein X-ray structures, were calculated using molecular dynamics simulations and MM-PBSA calculations. The alphaalpha conformations of five of the tetrapeptides were also studied. SALN has been earlier shown by molecular dynamics simulations and NMR spectroscopy to have a tendency to form an alpha(r)beta turn. The gas-phase energy of the molecular mechanical force field (CHARMM), the electrostatic and non-polar solvation free energies and solute entropies were used to explain the free energy differences of the alphaalpha, betabeta and alpha(r)beta conformations of the peptides. The alpha(r)beta conformation of SALN and SATN was predicted to be slightly more stable than the extended conformation (betabeta), in agreement with experimental results. The SALN mutants SAIN, SAVN, SATN, SSIN and MSHV, were also predicted to be potential alpha(r)beta turn-forming peptides. We report also revised positional potentials for the type VIII turn, based on a non-homologous set of protein structures. This protein databank analysis confirms the main results of the earlier analyses and reveals several new amino acid residues with a significant positional preference. The results of this work led us to suggest that the alpha(r)beta turn may be the most common turn type in peptides. Such turns may be readily formed in aqueous solution and thereby play important roles in the protein folding process by serving as an initiation point for structure formation.     

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like beta-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "beta-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the beta-strands in the beta-barrel of the fragments. The native-like beta-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two beta-turn regions, I-18-D-21 and Y-27-Q-30, and developed by the formation of a nonlocal nucleation site at the beta-barrel region. The formation of beta-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.     

Roles of beta-turns in protein folding: from peptide models to protein engineering

Reverse turns are a major class of protein secondary structure; they represent sites of chain reversal and thus sites where the globular character of a protein is created. It has been speculated for many years that turns may nucleate the formation of structure in protein folding, as their propensity to occur will favor the approximation of their flanking regions and their general tendency to be hydrophilic will favor their disposition at the solvent-accessible surface. Reverse turns are local features, and it is therefore not surprising that their structural properties have been extensively studied using peptide models. In this article, we review research on peptide models of turns to test the hypothesis that the propensities of turns to form in short peptides will relate to the roles of corresponding sequences in protein folding. Turns with significant stability as isolated entities should actively promote the folding of a protein, and by contrast, turn sequences that merely allow the chain to adopt conformations required for chain reversal are predicted to be passive in the folding mechanism. We discuss results of protein engineering studies of the roles of turn residues in folding mechanisms. Factors that correlate with the importance of turns in folding indeed include their intrinsic stability, as well as their topological context and their participation in hydrophobic networks within the protein's structure.     

Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c).

The crystal structures of four peptides incorporating 1-aminocycloheptane-1-carboxylic acid (Ac7c) are described. Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe adopt beta-turn conformations stabilized by an intramolecular 4----1 hydrogen bond, the former folding into a type-I/III beta-turn and the latter into a type-II beta-turn. In the dipeptide esters, Boc-Aib-Ac7c-OMe and Boc-Pro-Ac7c-OMe, the Ac7c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac7c-OMe found in the asymmetric unit. The cycloheptane ring of Ac7c residues adopts a twist-chair conformation in all the peptides studied. 1H-NMR studies in CDCl3 and (CD3)2SO and IR studies in CDCl3 suggest that Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe maintain the beta-turn conformations in solution.     

Reduced tendency to form a beta turn in peptides from the P22 tailspike protein correlates with a temperature-sensitive folding defect

A family of mutants of the P22 bacteriophage tailspike protein has been characterized as temperature sensitive for folding (tsf) by King and co-workers [King, J. (1986) Bio/Technology 4, 297-303]. There is substantial evidence that the tsf mutations alter the folding pathway but not the stability of the final folded protein. Several point mutations are known to cause the tsf phenotype; most of these occur in regions of the tailspike sequence likely to take up reverse turns. Hence, it has been hypothesized that the correct folding of the P22 tailspike protein requires formation of turns and that the mutations causing tsf phenotypes interfere at this critical stage. We have tested this hypothesis by study of isolated peptides corresponding to a region of the P22 tailspike harboring a tsf mutation. Comparison of the tendencies of wild-type and tsf sequences to adopt turn conformations was achieved by the synthesis of peptides with flanking cysteine residues and the use of a thiol-disulfide exchange assay. We find that the wild-type sequence, either as a decapeptide (Ac-CVKFPGIETC-CONH2) or as a dodecapeptide (Ac-CYVKFPGIETLC-CONH2), has a 3-5-fold greater tendency for its termini to approach closely enough to form the intramolecular disulfide than do the peptide sequences corresponding to the tsf mutant sequences, which have a Gly----Arg substitution (Ac-CVKFPRIETC-CONH2 or Ac-CYVKFPRIETLC-CONH2). A peptide with a D-Arg substituted for the Gly has a slightly higher turn propensity than does the wild type. Together with data from nuclear magnetic resonance analysis of the oxidized peptides, this suggests that a type II beta turn is favored by the wild-type sequence. Our results on isolated peptides from the P22 tailspike protein support the model for its folding that includes reverse turn formation as a critical step.     

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.