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Tobin R Sosnick

Publications and source records attributed to Tobin R Sosnick.

33 records · Page 2Linked to original sources
PubMed2004

Random-coil behavior and the dimensions of chemically unfolded proteins.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

Guanidine↗
PubMed2004

Early collapse is not an obligate step in protein folding.

The dimensions and secondary structure content of two proteins which fold in a two-state manner are measured within milliseconds of denaturant dilution using synchrotron-based, stopped-flow small-angle X-ray scattering and far-UV circular dichroism spectroscopy. Even upon a jump to strongly native conditions, neither ubiquitin nor common-type acylphosphatase contract prior to the major folding event. Circular dichroism and fluorescence indicate that negligible amounts of secondary and tertiary structures form in the burst phase. Thus, for these two denatured states, collapse and secondary structure formation are not energetically downhill processes even under aqueous, low-denaturant conditions. In addition, water appears to be as good a solvent as that with high concentrations of denaturant, when considering the over-all dimensions of the denatured state. However, the removal of denaturant does subtly alter the distribution of backbone dihedral phi,psi angles, most likely resulting in a shift from the polyproline II region to the helical region of the Ramachandran map. We consider the thermodynamic origins of these behaviors along with implications for folding mechanisms and computer simulations thereof.

Acid Anhydride Hydrolases↗
PubMed2004

Discerning the structure and energy of multiple transition states in protein folding using psi-analysis.

We quantify the degree to which folding occurs along a complex landscape with structurally distinct pathways using psi-analysis in combination with a protein engineering method that identifies native, non-covalent polypeptide interactions and their relative populations at the rate-limiting step. By probing the proximity of two specific partners, this method is extremely well-suited for comparison to theoretical simulations. Using ubiquitin as a model system, we detect individual pathways with site-resolved resolution, demonstrating that the protein folds through a native-like transition state ensemble with a common nucleus that contains heterogeneous features on its periphery. The consensus transition state topology has part of the major helix docked against four properly aligned beta-strands. However, structural heterogeneity exists in the transition state ensemble, wherein peripheral regions are differentially populated according to their relative stability. Pathway diversity reflects the variable order of formation of these peripheral elements, which radiate outward from the common nucleus. These results, which show only moderate agreement with traditional mutational phi-analysis, provide an extraordinarily detailed and quantitative description of protein folding.

Amino Acid Sequence↗
PubMed2004

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme.

The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I(eq)-to-N, and focused on the I(eq)-to-N transition. The present study focuses on the U-to-I(eq) transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5' end and Cy3 at the 3' end show that modifications required for labeling do not interfere with folding and help to define the Mg(2+) concentration range for the U-to-I(eq) transition. Histogram analysis of the Mg(2+)-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg(2+) concentrations on the time scale of seconds. The trajectories at intermediate Mg(2+) concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of "nonsudden jump" FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many "local" conformational switches. A free energy contour is constructed to illustrate the complex folding surface.

Circular Dichroism↗
PubMed2003

Investigations into sequence and conformational dependence of backbone entropy, inter-basin dynamics and the Flory isolated-pair hypothesis for peptides.

The populations and transitions between Ramachandran basins are studied for combinations of the standard 20 amino acids in monomers, dimers and trimers using an implicit solvent Langevin dynamics algorithm and employing seven commonly used force-fields. Both the basin populations and inter-conversion rates are influenced by the nearest neighbor's conformation and identity, contrary to the Flory isolated-pair hypothesis. This conclusion is robust to the choice of force-field, even though the use of different force-fields produces large variations in the populations and inter-conversion rates between the dominant helical, extended beta, and polyproline II basins. The computed variation of conformational and dynamical properties with different force-fields exceeds the difference between explicit and implicit solvent calculations using the same force-field. For all force-fields, the inter-basin transitions exhibit a directional dependence, with most transitions going through extended beta conformation, even when it is the least populated basin. The implications of these results are discussed in the context of estimates for the backbone entropy of single residues, and for the ability of all-atom simulations to reproduce experimental protein folding data.

Algorithms↗
PubMed2003

Large-scale context in protein folding: villin headpiece.

The villin headpiece folds autonomously in vitro forming three alpha-helical regions. Local propensities, however, strongly disfavor the formation of the C-terminal helix because most native residue pairs in that helix are hydrophobic/polar mismatches. Even the N-terminal helix is unfavored according to the AGADIR criterion. Our coarse-grained ab initio simulations reveal three-body correlations in which hydrophobic residues position to protect amide-carbonyl hydrogen bonds from attack by water, thus inducing the growth of the C-terminal helix and guiding the folding process. Similar correlations are also found in all-atom simulations with an implicit solvent model that accurately reproduces the results of simulations with explicit solvent molecules. The correlations establish a large-scale, many-body context that may be probed experimentally by introducing mutations of certain nonobvious residues that reside outside the native hydrophobic core but that are predicted to affect the folding rates and dynamics dramatically.

Alanine↗
PubMed2003

RNA folding: models and perspectives.

Intrinsic events during RNA folding include conformational search and metal ion binding. Several experimentally testable models have been proposed to explain how large ribozymes accomplish folding. Future challenges include the validation of these models, and the correlation of experimental results and theoretical simulations.

Kinetics↗
PubMed2002

D/H amide isotope effect in model alpha-helical peptides.

The contribution of amide related hydrogen bonds to protein stability has recently been evaluated using the "Cm experiment", which measures the D/H amide isotope effect in proteins. We show here using isolated alpha-helical peptides that there is a significant effect of denaturant concentration on the measured D/H isotope effect, and that valid comparison of different proteins requires correcting for differences in denaturant (GdmHCl) concentration. Finally our results suggest that H-bonds in an isolated alpha-helix may contribute more to helix stability because of less strain compared to those in helical proteins and that the buried helical H-bonds in helical proteins are not necessarily energetically more favorable than solvent exposed H-bonds in isolated helices.

Alanine↗
PubMed2002

Distinguishing foldable proteins from nonfolders: when and how do they differ?

When a denatured polypeptide is put into refolding conditions, it undergoes conformational changes on a variety of times scales. We set out here to distinguish the fast events that promote productive folding from other processes that may be generic to any non-folding polypeptide. We have apply an ab initio folding algorithm to model the folding of various proteins and their compositionally identical, random-sequence analogues. In the earliest stages, proteins and their scrambled-sequence counterparts undergo indistinguishable reductions in the extent to which they explore conformation space. For both polypeptides, an early contraction occurs but does not involve the formation of a distinct intermediate. Following this phase, however, the naturally-occurring sequences are distinguished by an increase in the formation of three-body correlations wherein a hydrophobic group desolvates and protects an intra-molecular hydrogen bond. These correlations are manifested in a mild but measurable reduction of the accessible configuration space beyond that of the random-sequence peptides, and portend the folding to the native structure. Hence, early events reflect a generic response of the denatured ensemble to a change in solvent condition, but the wild-type sequence develops additional correlations as its structure evolves that can reveal the protein's foldability.

Algorithms↗
PubMed2002

Dynamics of hydrogen bond desolvation in protein folding.

As proteins fold, a progressive structuring, immobilization and eventual exclusion of water surrounding backbone hydrogen bonds takes place. This process turns hydrogen bonds into major determinants of the folding pathway and compensates for the penalty of desolvation of the backbone polar groups. Taken as an average over all hydrogen bonds in a native fold, this extent of protection is found to be nearly ubiquitous. It is dynamically crucial, determining a constraint in the long-time limit behavior of coarse-grained ab initio simulations. Furthermore, an examination of one of the longest available (1micros) all-atom simulations with explicit solvent reveals that this average extent of protection is a constant of motion for the folding trajectory. We propose how such a stabilization is best achieved by clustering five hydrophobes around the backbone hydrogen bonds, an arrangement that yields the optimal stabilization. Our results support and clarify the view that hydrophobic surface burial should be commensurate with hydrogen-bond formation and enable us to define a basic desolvation motif inherent to structure and folding dynamics.

Algorithms↗
PubMed2002

Entropic benefit of a cross-link in protein association.

We introduce a method to estimate the loss of configurational entropy upon insertion of a cross-link to a dimeric system. First, a clear distinction is established between the loss of entropy upon tethering and binding, two quantities that are often considered to be equivalent. By comparing the probability distribution of the center-to-center distances for untethered and cross-linked versions, we are able to calculate the loss of translational entropy upon cross-linking. The distribution function for the untethered helices is calculated from the probability that a given helix is closer to its partner than to all other helices, the "Nearest Neighbor" method. This method requires no assumptions about the nature of the solvent, and hence resolves difficulties normally associated with calculations for systems in liquids. Analysis of the restriction of angular freedom upon tethering indicates that the loss of rotational entropy is negligible. The method is applied in the context of the folding of a ten turn helical coiled coil with the tether modeled as a Gaussian chain or a flexible amino acid chain. After correcting for loop closure entropy in the docked state, we estimate the introduction of a six-residue tether in the coiled coil results in an effective concentration of the chain to be about 4 or 100 mM, depending upon whether the helices are denatured or pre-folded prior to their association. Thus, tethering results in significant stabilization for systems with millimolar or stronger dissociation constants.

Dimerization↗
PubMed2002

Contribution of hydrogen bonding to protein stability estimated from isotope effects.

An unresolved issue in structural biology concerns the relative contribution of H bonds to protein stability. We use the small molecules 4-acetamidobenzoic acid and N-acetylanthranilic acid as model compounds to relate the energetic contribution from hydrogen bonds (H bonds) to the deuterium/hydrogen amide isotope effect. N-Acetylanthranilic acid models carbonyl-amide H bonds formed during protein folding; 4-acetamidobenzoic acid models the unfolded state in which the amide H bonds to water. NMR is used to measure shifts in the pK(a) of the ionizable carboxyl group when the amides of the compounds are either protonated or deuterated. From the pK(a) shift, we obtain a quantitative scale factor: SF = partial partial differential(DeltaG(HB))/partial partial differential(RT ln Phi), where DeltaG(HB) is the change in free energy of an H bond upon isotope substitution and Phi is the fractionation factor. Isotope effect data also are reported for a small globular protein, lambda repressor, using the "C(m) experiment". The protein's isotope effect, which reports on the shape of the energy well, is converted to H-bonding free energy by applying the scale factor. We estimate that amide-related H bonds (amide-carbonyl and amide-water) contribute favorably to protein stability by approximately 30-50 kcal/mol in lambda repressor, GCN4 coiled coil, and cytochrome c but unfavorably by approximately 6 kcal/mol in ubiquitin. The results indicate that H-bond strength varies from one protein to another and presumably at different sites within the same protein.

Amides↗
PubMed2002

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects.

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.

Acid Anhydride Hydrolases↗