Abstract: Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation–condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.

Abstract: I. Introduction 388 II. Case for Radicals 389 III. Radiation and Discharge Radical Sources 390 IV. Reaction Products: Nature of Amino Acid Modifications 391 V. Protein Integrity Versus Damage 392 VI. Reaction Kinetics and Protein Structure Elucidation 394 VII. Protein Folding: Equilibrium and Time-Resolved Studies 395 VIII. Protein Complexes and Assemblies 397 IX. Conclusions 398 Acknowledgments 399 References 399 This review describes mass spectrometry-based strategies and investigations to determine protein structure, folding dynamics, and protein-protein interactions in solution through the use of radical reagents. The radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources, and react with proteins on time scales that are less than those often attributed to structural reorganization and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt dynamic structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein–protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin and the binding of gelsolin to actin are among the systems presented to illustrate the approach. © 2002 Wiley Periodicals, Inc., Mass Spec Rev 20:388–401, 2001; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10013

Abstract: The oxidative folding of proteins consists of conformational folding and disulfide-bond reactions. These two processes are coupled significantly in folding-coupled regeneration steps, in which a single chemical reaction (the "forward" reaction) converts a conformationally unstable precursor species into a conformationally stable, disulfide-protected successor species. Two limiting-case mechanisms for folding- coupled regeneration steps are described. In the folded-precursor mechanism, the precursor species is preferentially folded at the moment of the forward reaction. The (transient) native structure increases the effective concentrations of the reactive thiol and disulfide groups, thus favoring the forward reaction. By contrast, in the quasi-stochastic mechanism, the forward reaction occurs quasi-stochastically in an unfolded precursor; i.e., reactive groups encounter each other with a probability determined primarily by loop entropy, albeit modified by conformational biases in the unfolded state. The resulting successor species is initially unfolded, and its folding competes with backward chemical reactions to the unfolded precursors. The folded-precursor and quasi-stochastic mechanisms may be distinguished experimentally by the dependence of their kinetics on factors affecting the rates of thiol -disulfide exchange and conformational (un)folding. Experimental data and structural and biochemical arguments suggest that the quasi-stochastic mechanism is more plausible than the folded-precursor mechanism for most proteins.

Abstract: Ultrafast-folding proteins are important for combining experiment and simulation to give complete descriptions of folding pathways. The WW domain family comprises small proteins with a three-stranded antiparallel β-sheet topology. Previous studies on the 57-residue YAP 65 WW domain indicate the presence of residual structure in the chemically denatured state. Here we analyze three minimal core WW domains of 38–44 residues. There was little spectroscopic or thermodynamic evidence for residual structure in either their chemically or thermally denatured states. Folding and unfolding kinetics, studied by using rapid temperature-jump and continuous-flow techniques, show that each domain folds and unfolds very rapidly in a two-state transition through a highly compact transition state. Folding half-times were as short as 17 μs at 25°C, within an order of magnitude of the predicted maximal rate of loop formation. The small size and topological simplicity of these domains, in conjunction with their very rapid two-state folding, may allow us to reduce the difference in time scale between experiment and theoretical simulation.

Abstract: Ribonuclease P (RNase P) is the endoribonuclease responsible for the 5'-maturation of precursor tRNA transcripts In bacteria, RNase P is composed of a catalytic RNA subunit and an associated protein subunit that enhances the substrate specificity of the holoenzyme We have initiated a study of the biophysical properties of the protein subunit from Bacillus subtilis RNase P (P protein) toward the goal of understanding the thermodynamics of RNase P holoenzyme assembly The P protein is predominantly unfolded in 10 mM sodium cacodylate at neutral pH based on circular dichroism and NMR studies and therefore has several characteristics typical of "intrinsically unstructured" proteins Furthermore, the P protein folds to its native R/‚ structure upon addition of various small molecule anions Anion-induced folding is best attributed to the binding of these anions to the folded state of the protein, and a model is presented which describes the observed tightly coupled folding and binding phenomena The P protein also undergoes a cooperative folding transition upon addition of the osmolyte trimethylamine N-oxide (TMAO) The equilibrium constant of folding (Kfold )a t 37°C for the P protein was determined to be 00071 ( 00005 using a two-state folding model to describe the TMAO titration data Thus, the folding and binding equilibria observed in the anion-induced folding of the P protein can be uncoupled to determine the intrinsic binding affinities ( Ka's) of the anionic ligands Evidence that the osmolyte-induced and the ligand-induced folded conformations of the P protein are structurally similar is also presented

Abstract: Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg 21 -induced tertiary folding of pyrene-labeled P4-P6. At 35 8C with [Mg 21 ] ’ 10 mM, P4-P6 folds on the tens of milliseconds timescale with kobs 5 15‐31 s ‐1 . From these values, an activation free energy DG ‡ of ;8‐16 kcal/mol is calculated, where the large range for DG ‡ arises from uncertainty in the preexponential factor relating kobs and DG ‡ . The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes F 5 DDG ‡ /DDG89 are ’0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The kobs depends on the Mg 21 concentration, and the initial slope of kobs versus [Mg 21 ] suggests that only ; 1M g 2 1 ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg 21 ions. The observation of a substantial DG ‡ despite an early folding transition state suggests that a simple two-state folding diagram for Mg 21 -induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.

Abstract: 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 ∼25% of the residues are involved in residual structures. Conformational shifts of the α-protons, heteronuclear 15N-{1H} NOEs and 15N 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 1H 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.

Abstract: Here we review different aspects of the protein folding literature. We present a broad range of observations, showing them to be consistent with a general hierarchical protein folding model. In such a model, local relatively stable, confor- mationally fluctuating building blocks bind through population selection, to yield the native state. The model includes several components: (1) the fluctuating build- ing blocks that constitute local minima along the polypeptide chain, which even if unstable still possess higher population times than all alternate conformations; (2) the landscape around the bottom of the funnels; (3) the consideration that protein folding involves intramolecular recognition; (4) similar landscapes are observed for folding and for binding, and that (5) the landscape is dynamic, changing with the conditions. The model considers protein folding to be guided by native inter- actions. The reviewed literature includes the effects of changing the conditions, intermediates and kinetic traps, mutations, similar topologies, fragment comple- mentation experiments, fragments and pathways, focusing on one specific well- studied example, that of the dihydrofolate reductase, chaperones, and chaperonines, in vivo vs. in vitro folding, still using the dihydrofolate example, amyloid forma- tion, and molecular "disorder". These are consistent with the view that binding and folding are similar events, with the differences stemming from different stabilities and hence population times.

Abstract: The flexibility and dynamics of proteins directly influence the processes of protein folding, recognition, and function. NMR spin relaxation methods are used to assess the dynamics and mobility of proteins, for fast ps and ns motions as well as slower μs and ms events. The degree of protein flexibility and disorder as well as the changes in protein flexibility can be assessed by NMR spin relaxation methods at individual residues within the protein. In addition to probing protein dynamics, the changes in the NMR-derived order parameters can be used to estimate the entropic contributions of order–disorder transitions. Furthermore, kinetic processes in the ms time regime may be directly investigated to extract the rates of conformational interconversion, ligand binding, and protein folding processes. We show how a variety of dynamical information can be obtained from NMR relaxation measurements. We present examples that illustrate the use of NMR spin relaxation analysis for investigation of folding and disorder in proteins. © 2001 by Elsevier Science Inc.

Abstract: We propose a coarse-grained model of proteins that take into account solvent effects and apply it for simulating folding of a three-helix-bundle protein. The energy functional form, refined from our previous work (Takada et al., J Chem Phys 1999;110:11616-11629), tries to closely imitate real physico-chemical interactions. In particular, the hydrogen bond that depends on local dielectric constant, the helix capping effect, and side-chain entropic effects are included. With use of the model, we simulate folding of the GA module of an albumin binding domain, 1prb(7-53), finding most trajectories reach at the native topology within 1 micros. In the simulation, helices 1 and 3 are mostly formed earlier accompanied by non-specific collapse, while second helix is intrinsically less stable and is formed with the help of tertiary contacts at later stage. We compute an analog of the transition state ensemble and compare it with those of other three-helix-bundle proteins. The transition state of 1prb(7-53) includes a few specific tertiary contacts of C terminus of helix 3 with the loop region between helices 1 and 2. This resembles, but is not equivalent to, an early formed region of fragment B of staphylococcal protein A, but is quite different from the folding transient structures of a de novo designed three-helix-bundle peptide.

Abstract: The thermodynamics of the native↔A state and native↔unfolded transitions for ubiquitin have been characterized in detail using the denaturants methanol and guanidinium chloride (Gdn·HCl) both separately and in combination. Gdn·HCl destabilizes the partially folded alcohol-induced A state such that the effects of alcoholic solvents on the native↔unfolded transition can be investigated directly via a two-state model. The combined denaturing effects of methanol and Gdn·HCl appear to conform to a simple additive model. We show that ubiquitin folds and unfolds cooperatively in all cases, forming the same “native” state; however, the thermodynamics of the N↔U transition change dramatically between alcoholic and Gdn·HCl solutions, with folding in aqueous methanol associated with large negative enthalpy and entropy terms at 298 K with a gradual falloff in ΔCp at higher methanol concentrations, as previously reported for the N↔A transition (Woolfson, D. N., Cooper, A., Harding, M. M., Williams, D. H., and Evans, P...

Abstract: Amino acid replacements were used to probe the roles of 14 sites in two well-characterized intermediates in the folding pathway of bovine pancreatic trypsin inhibitor (BPTI). One of these intermediates contains one of the three disulfides found in the native protein (30–51). NMR studies have shown that approximately two-thirds of this polypeptide has a native-like conformation. The other intermediate contains two native disulfides (30–51 and 5–55) and has a fully folded conformation. The φ-values for a majority of residues were <1, indicating that the native protein was significantly more destabilized than either intermediate even when the altered residue was located in a well-ordered region of the intermediate. These observations suggest that folding intermediates and transition states may generally be more structured than indicated by φ-values alone.

Abstract: Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.

Abstract: Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.

Abstract: The construction of a single-chain protein by linking the C terminal of one subunit with the N terminal of another in an otherwise dimeric protein has been considered as a strategy for increasing protein stability. 1,2 In this Communication we investigate the role of the covalent linkage in the stability of the folded protein and the implication for the folding kinetics. The class of dimeric proteins under consideration have hydrophobic cores formed by side chains from both subunits, such that they become unfolded when the subunits dissociate. In addition, the covalent linkage is assumed to be flexible and not form specific contacts with the rest of the single-chain protein. In this class are a number of well-characterized proteins, including the gene V dimer of bacteriophage f1, 1 the Arc repressor of bacteriophage P22, 2 and the coiled-coil region of the yeast transcription factor GCN4. 3

Abstract: We propose a coarse-grained lattice model for Monte Carlo simulations of folding of proteins consisting of several alpha-helices. A chain representing a protein is considered to contain A and B monomers forming relatively stiff A subchains, mimicking helices, and flexible B links between these subchains, respectively. Using this model, we simulate (1) folding of four-helix proteins in solution; (2) folding of membrane proteins containing one, two, or four helices; and (3) refolding of four-helix proteins adsorbed at the liquid-solid interface. For these cases, we show typical scenarios of protein folding and refolding and study the dependence of the folding time on the chain length. Combining the latter results with those already available in the literature, we discuss the relative rates of folding of proteins belonging to different classes.