Random Coil Behavior Research Articles

Denatured State Conformational Biases in Three-Helix Bundles Containing Divergent Sequences Localize near Turns and Helix Capping Residues.

Rhodopseudomonas palustris cytochrome c', a four-helix bundle, and the second ubiquitin-associated domain, UBA(2), a three-helix bundle from the human homologue of yeast Rad23, HHR23A, deviate from random coil behavior under denaturing conditions in a fold-specific manner. The random coil deviations in each of these folds occur near interhelical turns and loops in their tertiary structures. Here, we examine an additional three-helix bundle with an identical fold to UBA(2), but a highly divergent sequence, the first ubiquitin-associated domain, UBA(1), of HHR23A. We use histidine-heme loop formation methods, employing eight single histidine variants, to probe for denatured state conformational bias of a UBA(1) domain fused to the N-terminus of iso-1-cytochrome c (iso-1-Cytc). Guanidine hydrochloride (GuHCl) denaturation shows that the iso-1-Cytc domain unfolds first, followed by the UBA(1) domain. Denatured state (4 and 6 M GuHCl) histidine-heme loop formation studies show that as the size of the histidine-heme loop increases, loop stability decreases, as expected for the Jacobson-Stockmayer relationship. However, loops formed with His35, His31, and His15, of UBA(1), are 0.6-1.1 kcal/mol more stable than expected from the Jacobson-Stockmayer relationship, confirming the importance of deviations of the denatured state from random coil behavior near interhelical turns of helical domains for facilitating folding to the correct topology. For UBA(1) and UBA(2), hydrophobic clusters on either side of the turns partially explain deviations from random coil behavior; however, helix capping also appears to be important.

Cross-reactivity of Haemophilus influenzae type a and b polysaccharides: molecular modeling and conjugate immunogenicity studies.

Haemophilus influenzae is a leading cause of meningitis disease and mortality, particularly in young children. Since the introduction of a licensed conjugate vaccine (targeting the outer capsular polysaccharide) against the most prevalent serotype, Haemophilus influenzae serotype b, the epidemiology of the disease has changed and Haemophilus influenzae serotype a is on the rise, especially in Indigenous North American populations. Here we apply molecular modeling to explore the preferred conformations of the serotype a and b capsular polysaccharides as well as a modified hydrolysis resistant serotype b polysaccharide. Although both serotype b and the modified serotype b have similar random coil behavior, our simulations reveal some differences in the polysaccharide conformations and surfaces which may impact antibody cross-reactivity between these two antigens. Importantly, we find significant conformational differences between the serotype a and b polysaccharides, indicating a potential lack of cross-reactivity that is corroborated by immunological data showing little recognition or killing between heterologous serotypes. These findings support the current development of a serotype a conjugate vaccine.

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.

Moisture diffusivity in concentrated and dry protein-carbohydrate films

Understanding moisture diffusivity behaviour over a wide range of moisture contents is pivotal for optimising drying operations. Generally, data on moisture diffusivity are scarce and the effect of matrix composition on moisture diffusivity at relevant temperature for drying processes is not yet well described. In this paper moisture diffusivity in protein-carbohydrate films is systematically investigated for a wide range of moisture contents at 80 °C.Diffusion data are obtained from controlled thin film drying experiments following the regular regime method and compared to theoretical models. Moisture diffusivity for binary maltodextrin-water and whey protein-water systems appeared similar and were reasonably well described with the Darken relation. Diffusivity was lower for casein-water systems at moisture contents above 0.15 kg water/kg, which may be explained by compartmentalization of water in the casein micelles. At low moisture contents all binary systems showed universal behaviour, which may be explained by random coil behaviour leading to similar water-molecule interactions. This behaviour could be well described by free-volume theory. In mixed systems of proteins and carbohydrates moisture diffusivity appeared strongly influenced by the presence of casein, probably due to their high voluminosity. Finally, it was surprisingly observed that diffusivity in multicomponent systems decreased sharply at lower water contents when compared to binary systems. This might be explained by a denser molecular packed system in the dry regime for multicomponent systems or water trapping by protein-carbohydrate complexes.

NMR characterization of solvent accessibility and transient structure in intrinsically disordered proteins

In order to understand the conformational behavior of intrinsically disordered proteins (IDPs) and their biological interaction networks, the detection of residual structure and long-range interactions is required. However, the large number of degrees of conformational freedom of disordered proteins require the integration of extensive sets of experimental data, which are difficult to obtain. Here, we provide a straightforward approach for the detection of residual structure and long-range interactions in IDPs under near-native conditions using solvent paramagnetic relaxation enhancement (sPRE). Our data indicate that for the general case of an unfolded chain, with a local flexibility described by the overwhelming majority of available combinations, sPREs of non-exchangeable protons can be accurately predicted through an ensemble-based fragment approach. We show for the disordered protein α-synuclein and disordered regions of the proteins FOXO4 and p53 that deviation from random coil behavior can be interpreted in terms of intrinsic propensity to populate local structure in interaction sites of these proteins and to adopt transient long-range structure. The presented modification-free approach promises to be applicable to study conformational dynamics of IDPs and other dynamic biomolecules in an integrative approach.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.

Probing Denatured State Conformational Bias in a Three-Helix Bundle, UBA(2), Using a Cytochrome c Fusion Protein.

Previous work with the four-helix-bundle protein cytochrome c' from Rhodopseudomonas palustris using histidine-heme loop formation methods revealed fold-specific deviations from random coil behavior in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix-bundle polypeptide, the second ubiquitin-associated domain, UBA(2), of the human DNA excision repair protein. We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain at the N-terminus of iso-1-cytochrome c. We have engineered histidine into highly solvent accessible positions of UBA(2), creating six single histidine variants. Guanidine hydrochloride denaturation studies show that the UBA(2)-cytochrome c fusion protein unfolds in a three-state process with iso-1-cytochrome c unfolding first. Furthermore, engineered histidine residues in UBA(2) strongly destabilize the iso-1-cytochrome c domain. Equilibrium and kinetic histidine-heme loop formation measurements in the denatured state at 4 and 6 M guanidine hydrochloride show that loop stability decreases as the size of the histidine-heme loop increases, in accord with the Jacobson-Stockmayer equation. However, we observe that the His27-heme loop is both more stable than expected from the Jacobson-Stockmayer relationship and breaks more slowly than expected. These results show that the sequence near His27, which is in the reverse turn between helices 2 and 3 of UBA(2), is prone to persistent interactions in the denatured state. Therefore, consistent with our results for cytochrome c', this reverse turn sequence may help to establish the topology of this fold by biasing the conformational distribution of the denatured state.

A Helix 1 Peptide from UBA(1) Provides Evidence for Tertiary Interactions in the Denatured State of UBA(1)

Studies over recent decades have shown that the denatured state ensemble (DSE) - the collection of all random-coil conformations of a denatured protein - exhibits significant conformational bias that deviates from theoretical random coil behavior. This inherent bias is thus integral to the initial stages of protein folding and can give insight into unstructured proteins, such as intrinsically disordered proteins (IDPs). In this work, we probe the denatured state of a three-helix bundle protein termed the ubiquitin-associated domain, UBA(1), derived from the DNA-excision repair protein HHR23A. UBA(1) is strongly denatured using guanidine hydrochloride (GdnHCl) and investigated using circular dichroism (CD) spectroscopy. Under moderate to strongly denaturing conditions, UBA(1) demonstrates persistent α-helical content, eventually forming significant polyproline-II (PII) helical structure at 7M GdnHCl. Isolation of the first helix of UBA(1) via insertion of a stop codon at R177 reveals substantially decreased per-residue helicity at all concentrations of denaturant when compared to full protein. This indicates that full-length UBA(1) maintains transient tertiary contacts under strongly denaturing conditions. We have also acquired high resolution NMR (15N-HSQC, HNCO, and HNCA) spectra of UBA(1) to obtain chemical shifts of the backbone atoms for each residue in denaturant. Our data provide residue-specific secondary structure information and indicate that UBA(1) forms transient native-like helices of nearly equal proportion in the DSE. These data will be compared to backbone chemical shifts of the isolated first helix of UBA(1) to further elucidate contributions of transient tertiary contacts to residual helical content in the first helix of UBA(1) in the DSE of full length UBA(1).

The polypeptide biophysics of proline/alanine-rich sequences (PAS): Recombinant biopolymers with PEG-like properties.

PAS polypeptides comprise long repetitive sequences of the small L‐amino acids proline, alanine and/or serine that were developed to expand the hydrodynamic volume of conjugated pharmaceuticals and prolong their plasma half‐life by retarding kidney filtration. Here, we have characterized the polymer properties both of the free polypeptides and in fusion with the biopharmaceutical IL‐1Ra. Data from size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and quantification of hydrodynamic and polar properties demonstrate that the biosynthetic PAS polypeptides exhibit random coil behavior in aqueous solution astonishingly similar to the chemical polymer poly‐ethylene glycol (PEG). The solvent‐exposed PAS peptide groups, in the absence of secondary structure, account for strong hydrophilicity, with negligible contribution by the Ser side chains. Notably, PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity. Their uniform monodisperse composition as genetically encoded polymers and their biological nature, offering biodegradability, render PAS polypeptides a promising PEG mimetic for biopharmaceutical applications.

The LC8 Recognition Motif Preferentially Samples Polyproline II Structure in Its Free State.

LC8 is a ubiquitous hub protein that binds intrinsically disordered proteins and promotes their assembly into higher-order complexes. A common feature among the more than 100 essential LC8 binding proteins is that in the 10-12-amino acid recognition sequence there is a conserved QT motif but variable amino acids N- and C-terminal to the QT pair. The sequence diversity among LC8 binding partners implies that structural factors also contribute to specificity. To investigate whether one such factor is the transient secondary structure favored by an LC8 binding sequence, we report here a molecular ensemble description of ICTL, a domain of the dynein intermediate chain that includes binding sites for light chains LC8 and Tctex1. Nuclear magnetic resonance secondary chemical shifts and residual dipolar coupling values combined with ensemble generation and selection algorithms indicate a deviation from statistical (random) coil behavior with an elevated population of polyproline II (PPII) conformations for the ICTL regions that bind LC8 and Tctex1. Independent measurements of one- and three-bond scalar couplings confirm the PPII transient secondary structure propensity. Given that in the IC/Tctex1/LC8 ternary complex ICTL forms a β-strand at the interface of Tctex1 and LC8, we hypothesize that a PPII conformation may facilitate its initial docking and insertion into the binding cleft of the β-sheet LC8 dimer interface. Molecular ensemble calculations for intrinsically disordered LC8 binding partners also reveal PPII conformational sampling within and proximate to the LC8 recognition motifs, suggesting that a preference for a PPII conformation is general for LC8 binding partners.

Random-Coil Behavior of Chemically Denatured Topologically Knotted Proteins Revealed by Small-Angle X-ray Scattering.

Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils. By revisiting the scaling law of radius of gyration (Rg) as a function of polypeptide chain length for chemically denatured proteins and natively folded proteins, we find that the chemically denatured knotted proteins in fact follow the same random coil-like behavior, suggesting that the formation of topological protein knots do not necessarily require global compaction while the loosely knotted polypeptide chains are capable of maintaining the correct chirality without defined secondary or tertiary structures.

How Do Interactions in Cis with Ordered Domains Influence Sequence-Ensemble Relationships of Intrinsically Disordered Regions?

The conformational properties of intrinsically disordered proteins (IDPs) are governed by their amino acid sequences. An overwhelming majority of disordered regions are often found in cis with ordered domains as terminal tails or inter-domain linkers. The motivating question is if the rules governing sequence-ensemble relationships that have been inferred for IDPs as autonomous units apply for describing the conformational properties of disordered regions in cis to ordered domains. Using atomistic simulations and fluorescence studies we characterize the conformational properties of several archetypes of intrinsically disordered sequences in the presence and absence of cis-acting ordered domains. In particular, we quantify the effects of sequence composition, chain length, and sequence patterning of disordered sequences on the inter-domain coupling. The balance between intra-domain and inter-domain interactions can modulate intrinsic conformational propensities of the disordered regions. The circumstances giving rise to convergence toward generic random-coil behavior for disordered regions in cis to ordered domains will be highlighted.

Why is Taq DNA Polymerase so Stable? A Reduced Entropic Folding Penalty and the Denatured State Ensemble

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases reveals that Klentaq's extreme free energy of folding originates from a significantly decreased entropic folding penalty. A parallel solution-based structural analysis reveals that the denatured state of the mesophilic polymerase (Klenow) is significantly more elongated than the denatured state of the thermophilic Taq polymerase and thus has a significantly larger radius of gyration. The combined data indicate that the folded state of the thermophilic polymerase is not more intrinsically stable: instead, its more globular denatured state has a considerably reduced entropic and structural barrier between the denatured and native states, resulting in a much more favorable ΔG of folding. The data also indicate that the stability-linked evolutionary differences between the two proteins are expressed primarily in the denatured state. This is the first direct structural demonstration of denatured state size/shape differences in a mesophilic-thermophilic protein pair. Despite their demonstrably different sizes and structures, both denatured state ensembles still fall within the range of random-coil behavior.

Effect of PEGylation on Protein Hydrodynamics

We studied the effect of PEGylation on protein hydrodynamic behavior using hen egg-white lysozyme (HEWL) as a model protein. HEWL was PEGylated with a linear, 20 kDa PEG using reductive amination to produce PEG1-, PEG2-, and PEG3-HEWL. Near- and far-UV-CD spectroscopy revealed no significant effect of PEGylation on HEWL higher order structure. SDS-PAGE, mass spectrometry, online static light scattering (SLS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were employed to characterize the heterogeneity and molecular weights of the purified PEG-HEWL molecules, the results of which underscored the importance of using first-principle based methods for such analyses along with the underlying complexities of characterizing PEG-protein conjugates. Hydrodynamic characterization of various linear and branched PEGs (5-40 kDa) and PEG-HEWL molecules was performed using dynamic light scattering (DLS) and SV-AUC. The PEG polymer exhibited a random-coil conformation in solution with the M(w) ∝ R(h)(n) scaling relationship yielding a scaling exponent (n) = 2.07. Singly branched PEGs were also observed to exhibit random-coil behavior with Stokes radii identical to those of their linear counterparts. SV-AUC studies of PEG-HEWL showed PEG has a "parachute" like effect on HEWL, and dramatically increases the frictional drag; PEG-HEWL also exhibited random-coil-like characteristics in solution (n = 1.8). The sedimentation coefficient (s) of PEG-HEWL remained invariant with increasing degree of PEGylation, indicating that the increase in molecular mass from PEG was compensated by an almost equivalent increase in frictional drag. Our studies draw caution to using SV-AUC for the characterization of size heterogeneity of PEG-protein mixtures.

Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, 15N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (Rh=25 Å) relative to the urea-denatured state (Rh≥30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'.

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.

Denatured states of low-complexity polypeptide sequences differ dramatically from those of foldable sequences

How the primary sequence of a protein encodes conformational preferences that operate early in folding to promote efficient formation of the correct native topology is still poorly understood. To address this issue, we have prepared a set of yeast iso-1-cytochrome c variants that contain polyalanine inserts ranging from 6 to 30 residues in length near the N terminus of the protein. We study the thermodynamics and kinetics of His-heme loop formation in the denatured state at 3 and 6 M guanidine-HCl concentration. We find that polyalanine closely approximates a random coil with excluded volume giving scaling exponents, nu(3), for equilibrium loop formation of 2.26 +/- 0.13 and 1.97 +/- 0.04 in 3 and 6 M guanidine-HCl, respectively. The rate of loop breakage initially decreases and then becomes independent of loop size as would be expected for a random coil. Comparison with previously reported data for denatured state His-heme loop formation for iso-1-cytochrome c and Rhodopseudomonas palustris cytochrome c', shows that foldable sequences deviate significantly from random coil behavior and that the deviation is fold-dependent.

15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide (15)N is used as a probe of solvent exchange. The experiment is performed in 50% H(2)O-50% D(2)O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, (15)N(D), into protonated species, (15)N(H), as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of (15)N(H/D)-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5-2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.

Thermodynamics of Loop Formation in the Denatured State of Rhodopseudomonas palustris Cytochrome c′: Scaling Exponents and the Reconciliation Problem

The observation that denatured proteins yield scaling exponents, ν, consistent with random-coil behavior and yet can also have pockets of residual or nonrandom structure has been termed the “reconciliation problem”. To provide greater insight into the denatured state of a foldable sequence, we have measured histidine–heme loop formation equilibria in the denatured state of a class II c-type cytochrome, cytochrome c′ from Rhodopseudomonas palustris. We have prepared a series of variants that provide His–heme loop stabilities, p K loop(His), for loop sizes ranging from 10 to 111 residues at intervals of 7 to 11 residues along the sequence of the protein. We observe a scaling exponent for loop formation, ν 3, of 2.5 ± 0.3. Theoretical values for ν 3 range from 1.8 to 2.4; thus, the observed ν 3 is consistent with random-coil behavior. However, in contrast to data for loop formation as a function of loop size obtained with peptides of homogeneous sequence, we observe considerable scatter about the linear dependence of loop stability on loop size. Thus, foldable sequences behave very differently from homogeneous peptide sequences. The observed scatter suggests that there is considerable variation in the conformational properties along the backbone of a foldable sequence, consistent with alternating compact and extended regions. With regard to the reconciliation problem, it is evident that a scaling exponent consistent with a random coil is necessary but not sufficient to demonstrate random-coil behavior.

Thermodynamic and Structural Origins for the Thermostability of Taq DNA Polymerase

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. We have previously shown that at 25oC, Klentaq polymerase (the large fragment domain of Taq) has a folding free energy (δGfold) of ‐27 kcal/mole, making it one of the most stable monomeric proteins yet characterized. In contrast, Klenow polymerase has a δGfold of only ‐4.7 kcal/mole at 25oC, which is quite low for a protein of its size (68kDa). Here we report full stability curves (δG versus temperature) for Klentaq and Klenow, which reveal that Klentaq's extreme free energy of folding originates from a significantly decreased entropic penalty of folding (δSconf). Structural analyses of the denatured‐state ensembles for the two proteins indicate that the source of these energetics is a significantly smaller and less solvent accessible denatured state for Klentaq relative to Klenow. It is also notable that although the average denatured state diameter for Klenow is approximately 1.4X larger than for Klentaq, they both fall within the range for random‐coil behavior.