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Helix-coil models are routinely used to interpret circular dichroism data of helical peptides or predict the helicity of naturally-occurring and designed polypeptides. However, a helix-coil model contains significantly more information than mean helicity alone, as it defines the entire ensemble-the equilibrium population of every possible helix-coil configuration-for a given sequence. Many desirable quantities of this ensemble are either not obtained as ensemble averages or are not available using standard helicity-averaging calculations. Enumeration of the entire ensemble can allow calculation of a wider set of ensemble properties, but the exponential size of the configuration space typically renders this intractable. We present an algorithm that efficiently approximates the helix-coil ensemble to arbitrary accuracy by sequentially generating a list of the M highest populated configurations in descending order of population. Truncating this list of (configuration, population) pairs at a desired accuracy provides an approximating sub-ensemble. We demonstrate several uses of this approach for providing insight into helix-coil ensembles and folding mechanisms, including landscape visualization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Histatin 5 (Hist5) is an antimicrobial peptide found in human saliva as part of the innate immune system. Hist5 can bind several metal ions in vitro, and Zn 2+ has been shown to function as an inhibitory switch to regulate the peptide's biological activity against the opportunistic fungal pathogen Candida albicans in cell culture. Here, we studied Zn 2+ binding to Hist5 at four temperatures from 15 to 37 °C using isothermal titration calorimetry to obtain thermodynamic parameters that were corrected for competing buffer effects. Hist5 bound Zn 2+ with a buffer-dependent association constant of ∼10 5 M -1 and a buffer-independent association constant of ∼6 × 10 6 M -1 at pH 7.4 and at all temperatures tested. Zn 2+ binding was both enthalpically and entropically favorable, with larger entropic contributions at 15 °C and larger enthalpic contributions at 37 °C. Additionally, the Zn:Hist5 binding stoichiometry increased from 1:1 to 2:1 as temperature increased. The enthalpy-entropy compensation and the variable stoichiometry lead us to propose a model in which the Zn-Hist5 complex exists in an equilibrium between two distinct binding modes with different Zn:Hist5 stoichiometries. The in-depth thermodynamic analysis presented herein may help illuminate the biophysical basis for Zn-dependent changes in the antifungal activity of Hist5.

RNA recognition frequently results in conformational changes that optimize intermolecular binding. As a consequence, the overall binding affinity of RNA to its binding partners depends not only on the intermolecular interactions formed in the bound state but also on the energy cost associated with changing the RNA conformational distribution. Measuring these "conformational penalties" is, however, challenging because bound RNA conformations tend to have equilibrium populations in the absence of the binding partner that fall outside detection by conventional biophysical methods. In this study we employ as a model system HIV-1 TAR RNA and its interaction with the ligand argininamide (ARG), a mimic of TAR's cognate protein binding partner, the transactivator Tat. We use NMR chemical shift perturbations and relaxation dispersion in combination with Bayesian inference to develop a detailed thermodynamic model of coupled conformational change and ligand binding. Starting from a comprehensive 12-state model of the equilibrium, we estimate the energies of six distinct detectable thermodynamic states that are not accessible by currently available methods. Our approach identifies a minimum of four RNA intermediates that differ in terms of the TAR conformation and ARG occupancy. The dominant bound TAR conformation features two bound ARG ligands and has an equilibrium population in the absence of ARG that is below detection limit. Consequently, even though ARG binds to TAR with an apparent overall weak affinity ( K d app ≈ 0.2 mM), it binds the prefolded conformation with a K d in the nM range. Our results show that conformational penalties can be major determinants of RNA-ligand binding affinity as well as a source of binding cooperativity, with important implications for a predictive understanding of how RNA is recognized and for RNA-targeted drug discovery.

Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order-disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helices, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temperatures. Through coarse-grain simulations, we show that hysteresis arises from physical crosslinking due to mesoscale phase separation of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temperature, they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.

The flexibility of biological macromolecules is an important structural determinant of function. Unfortunately, the correlations between different motional modes are poorly captured by discrete ensemble representations. Here, we present new ways to both represent and visualize correlated interdomain motions. Interdomain motions are determined directly from residual dipolar couplings, represented as a continuous conformational distribution, and visualized using the disk-on-sphere representation. Using the disk-on-sphere representation, features of interdomain motions, including correlations, are intuitively visualized. The representation works especially well for multidomain systems with broad conformational distributions.This analysis also can be extended to multiple probability density modes, using a Bingham mixture model. We use this new paradigm to study the interdomain motions of staphylococcal protein A, which is a key virulence factor contributing to the pathogenicity of Staphylococcus aureus. We capture the smooth transitions between important states and demonstrate the utility of continuous distribution functions for computing the reorientational components of binding thermodynamics. Such insights allow for the dissection of the dynamic structural components of functionally important intermolecular interactions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1-1 s -1 , for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

The Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies. SpA includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner have apparently been unsuccessful. There have also been no previous structures of tandem domains. Here we report the high-resolution crystal structures of a single C domain, and of two B domains connected by the conserved linker. Both structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in side chain conformational networks and contacting protein interfaces. This represents the kind of structural plasticity that could enable SpA to bind multiple partners., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Staphylococcal protein A (SpA) is a multidomain protein consisting of five globular IgG binding domains separated by a conserved six- to nine-residue flexible linker. We collected SAXS data on the N-terminal protein-binding half of SpA (SpA-N) and constructs consisting of one to five domain modules in order to determine statistical conformation of this important S. aureus virulence factor. We fit the SAXS data to a scattering function based on a new polymer physics model, which provides an analytical description of the SpA-N statistical conformation. We describe a protocol for systematically determining the appropriate level of modeling to fit a SAXS data set based on goodness of fit and whether the addition of parameters improves it. In the case of SpA-N, the analytical polymer physics description provides a depiction of the statistical conformation of a flexible protein that, while lacking atomistic detail, properly reflects the information content of the data.

Coupled ligand binding and conformational change plays a central role in biological regulation. Ligands often regulate protein function by modulating conformational dynamics, yet the order in which binding and conformational change occurs are often hotly debated. Here we show that the "conformational selection versus induced fit" distinction on which this debate is based is a false dichotomy because the mechanism depends on ligand concentration. Using the binding of pyrophosphate (PPi) to Bacillus subtilis RNase P protein as a model, we show that coupled reactions are best understood as a change in flux between competing pathways with distinct orders of binding and conformational change. The degree of partitioning through each pathway depends strongly on PPi concentration, with ligand binding redistributing the conformational ensemble toward the folded state by both increasing folding rates and decreasing unfolding rates. These results indicate that ligand binding induces marked and varied changes in protein conformational dynamics, and that the order of binding and conformational change is ligand concentration dependent.

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics., (Copyright © 2010 Wiley-Liss, Inc.)

Protein folding intermediates are often imperative for overall folding processes and consequent biological functions. However, the low population and transient nature of the intermediate states often hinder their biochemical and biophysical characterization. Previous studies have demonstrated that Bacillus subtilis ribonuclease P protein (P protein) is conformationally heterogeneous and folds with multiphasic kinetics, indicating the presence of an equilibrium and kinetic intermediate in its folding mechanism. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to study the ensemble corresponding to this intermediate (I). The results indicate that the N-terminal and C-terminal helical regions are mostly unfolded in I. 1H−15N heteronuclear single-quantum coherence NMR spectra collected as a function of pH suggest that the protonation of His 22 may play a major role in the energetics of the equilibria among the unfolded, intermediate, and folded state ensembles of P protein. NMR paramagnetic relaxation enhancement experiments were also used to locate the small anion binding sites in both the intermediate and folded ensembles. The results for the folded protein are consistent with the previously modeled binding regions. These structural insights suggest a possible role for I in the RNase P holoenzyme assembly process.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the "induced fit" mechanism (binding first) or "conformational selection" (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a flux-based approach.

Interconversion of protein conformations is imperative to function, as evidenced by conformational changes associated with enzyme catalytic cycles, ligand binding and post-translational modifications. In this study, we used 15N NMR relaxation experiments to probe the fast (i.e., ps-ns) and slow (i.e., micros-ms) conformational dynamics of Bacillus subtilis ribonuclease P protein (P protein) in its folded state, bound to two sulfate anions. Using the Lipari-Szabo mapping method [Andrec, M., Montelione, G. T., and Levy, R. M. (2000) J. Biomol. NMR 18, 83-100] to interpret the data, we find evidence for P protein dynamics on the mus-ms time scale in the ensemble. The residues that exhibit these slow internal motions are found in regions that have been previously identified as part of the P protein-P RNA interface. These results suggest that structural flexibility within the P protein ensemble may be important for proper RNase P holoenzyme assembly and/or catalysis.

Analysis of biopolymer sequences and structures generally adopts one of two approaches: use of detailed biophysical theoretical models of the system with experimentally-determined parameters, or largely empirical statistical models obtained by extracting parameters from large datasets. In this work, we demonstrate a merger of these two approaches using Bayesian statistics. We adopt a common biophysical model for local protein folding and peptide configuration, the helix-coil model. The parameters of this model are estimated by statistical fitting to a large dataset, using prior distributions based on experimental data. L(1)-norm shrinkage priors are applied to induce sparsity among the estimated parameters, resulting in a significantly simplified model. Formal statistical procedures for evaluating support in the data for previously proposed model extensions are presented. We demonstrate the advantages of this approach including improved prediction accuracy and quantification of prediction uncertainty, and discuss opportunities for statistical design of experiments. Our approach yields a 39% improvement in mean-squared predictive error over the current best algorithm for this problem. In the process we also provide an efficient recursive algorithm for exact calculation of ensemble helicity including sidechain interactions, and derive an explicit relation between homo- and heteropolymer helix-coil theories and Markov chains and (non-standard) hidden Markov models respectively, which has not appeared in the literature previously.

beta-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that beta-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and beta-arrestin2, these studies are not representative of conformational changes in beta-arrestin1. Accordingly, in this study, we determine conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor (V(2)Rpp) or the corresponding unphosphorylated peptide (V(2)Rnp). V(2)Rpp binds specifically to beta-arrestin1 causing significant conformational changes, whereas V(2)Rnp does not alter the conformation of beta-arrestin1. Upon V(2)Rpp binding, we show that the previously shielded Arg(393) becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg(285) becomes more accessible, and this residue is located in a region of beta-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1, and we also show a functional consequence of the release of the C terminus of beta-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of beta-arrestin1 in the presence of V(2)Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in beta-arrestin1 when compared with beta-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.

Elastin-like polypeptides (ELPs) are stimulus-responsive polymers that contain repeats of five amino acids, Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. While studying the conformational mechanics of ELPs over a range of solvent conditions by single-molecule force spectroscopy, we noticed that some force-extension curves showed temperature-independent, extensional transitions that could not be fitted with a freely jointed chain or worm-like chain model. Here we show that the observed molecular elongation results from the force-induced peptidyl-prolyl cis-trans isomerization in prolines, which are repeated every fifth residue in the main chain of ELPs. Control experiments with poly(L-proline) demonstrate the similarity of the conformational transition between poly(L-proline) and ELPs. In contrast, the force-extension behavior of poly(L-lysine) showed no deviation in the relevant force range. Force-extension curves in hysteresis experiments showed an elongational difference between extension and relaxation pathways that suggests that the cis conformational state of the prolines could be exhausted on the time scale of the experiment. We present further computational evidence for this mechanism by Monte Carlo simulation of the force-extension behavior using an elastically coupled, two-state model. We believe ours is the first demonstration of force-induced prolyl cis-trans isomerization in proline-containing polypeptides. Our results suggest that single-molecule force spectroscopy could provide an alternate means to assay this important conformational transition in polypeptides.

Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.

The antibody binding properties of staphylococcal protein A (SpA) can be attributed to the presence of five highly homologous domains (E, D, A, B, and C). Although the folding of the B domain of protein A (BdpA) is well-characterized, the folding behavior of this domain in the context of full-length SpA in the cell remains unexplored. The sequence of the B domain is 89 and 91% identical to those of domains A and C, respectively. We have fused B domain sequences (BBdpA) as a close approximation of the A-B or B-C portion of SpA. Circular dichroism and fluorescence-detected denaturation curves of BBdpA are experimentally indistinguishable from those of BdpA. The rate constants for folding and unfolding from NMR line shape analysis for the single- and double-domain proteins are the same within experimental uncertainties (+/-20%). These results support the designation of SpA as a multiple independently-folding domain (MIFD) protein. We develop a mathematical model that describes the folding thermodynamics and kinetics of MIFD proteins. The model depicts MIFD protein folding and unfolding as a parallel network and explicitly calculates the flux through all parallel pathways. These fluxes are combined to give a complete description of the global thermodynamics and kinetics of the folding and unfolding of MIFD proteins. The global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein. We show that the global unfolding rate of a MIFD protein may be many orders of magnitude slower than that of the constituent domains.

Bacillus subtilis ribonuclease P protein (P protein) is predominantly unfolded (D) at physiological pH and low ionic strength; however, small molecule anionic ligands (e.g., sulfate) directly bind to and stabilize the folded state (NL2). Because the D + 2L <--> NL2 transition is experimentally two-state, high-energy states such as the singly bound, folded species (NL) and the unliganded folded species (N) are generally difficult to detect at equilibrium. To study the conformational properties of these ensembles, NMR-detected amide hydrogen exchange (HX) rates of P protein were measured at four sulfate (i.e., ligand) concentrations, a method we denote "ligation-state hydrogen exchange". The ligand concentration dependence of the HX rate of 47 residues was fit to a model with four possible HX pathways, corresponding to the local and/or global opening reactions from NL2 and NL, the local opening of N, and the global opening of N to D. Data analysis permits the calculation of the residue-specific free energy of opening from each ensemble as well as the fractional amide HX flux through each pathway. Results indicate that the predominant route of HX is through the NL and N states, which represent only 0.45% and 0.0005% of the total protein population in 20 mM sodium sulfate, respectively. Despite the low population of N, a region of protected amides was identified. Therefore, exchange through unliganded forms must be accounted for prior to the interpretation of HX-based protein-interaction studies. We offer a simple test to determine if HX occurs through the liganded or unliganded form.

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

In Bacillus subtilis, P protein is the noncatalytic component of ribonuclease P (RNase P) that is critical for achieving maximal nuclease activity under physiological conditions. P protein is predominantly unfolded (D) at neutral pH and low ionic strength; however, it folds upon the addition of sulfate anions (ligands) as well as the osmolyte trimethylamine N-oxide (TMAO) [Henkels, C. H., Kurz, J. C., Fierke, C. A., and Oas, T. G. (2001) Biochemistry 40, 2777-2789]. Since the molecular mechanisms that drive protein folding for these two solutes are different, CD thermal denaturation studies were employed to dissect the thermodynamics of protein unfolding from the two folded states. A global fit of the free-energy of TMAO-folded P protein versus [TMAO] and temperature yields T(S), DeltaH(S), and DeltaC(p) of unfolding for the poorly populated, unliganded, folded state (N) in the absence of TMAO. These thermodynamic parameters were used in the fit of the data from the coupled unfolding/ligand dissociation reaction to obtain the sulfate dissociation constant (K(d)) and the DeltaH and DeltaC(p) of dissociation. These fits yielded a DeltaC(p) of protein unfolding of 826 +/- 23 cal mol(-)(1) K(-)(1) and a DeltaC(p) of 1554 +/- 29 cal mol(-)(1) K(-)(1) for the coupled unfolding and dissociation reaction (NL(2) --> D + 2L). The apparent stoichiometry of sulfate binding is two, so the DeltaC(p) increment of ligand dissociation is 363 +/- 9 cal mol(-)(1) K(-)(1) per site. Because N and NL(2) appear to be structurally similar and therefore similarly solvated using standard biophysical analyses, we attribute a substantial portion of this DeltaC(p) increment to an increase in conformational heterogeneity coincident with the NL(2) --> N + 2L transition.

Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of alpha-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33; Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641]. We have made site-specific substitutions in the N- and C-cap motifs of the 5-helix protein monomeric lambda repressor (lambda(6-85)) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly context-dependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B-domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 microsec. Although wild-type BdpA folds extremely fast, simple-point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all-atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence-based time-resolved temperature-jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.

The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W*, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W*, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W* is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45 degrees C of 249,000 s(-1). Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least approximately 2 x 10(6) s(-1).

The effects of amino acid replacements on the backbone dynamics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments. Previous studies have shown that backbone amide groups within the trypsin-binding region of the wild-type protein undergo conformational exchange processes on the micros time scale, and that replacement of Tyr35 with Gly greatly increases the number of backbone atoms involved in such motions. In order to determine whether these mutational effects are specific to the replacement of this residue with Gly, six additional replacements were examined in the present study. In two of these, Tyr35 was replaced with either Ala or Leu, and the other four were single replacements of Tyr23, Phe33, Asn43 or Asn44, all of which are highly buried in the native structure and conserved in homologous proteins. The Y35A and Y35L mutants displayed dynamic properties very similar to those of the Y35G mutant, with the backbone segments including residues 10-19 and 32-44 undergoing motions revealed by enhanced 15N transverse relaxation rates. On the other hand, the Y23L, N43G and N44A substitutions caused almost no detectable changes in backbone dynamics, on either the ns-ps or ms-micros time scales, even though each of these replacements significantly destabilizes the native conformation. Replacement of Phe33 with Leu caused intermediate effects, with several residues that have previously been implicated in motions in the wild-type protein displaying enhanced transverse relaxation rates. These results demonstrate that destabilizing amino acid replacements can be accommodated in a native protein with dramatically different effects on conformational dynamics and that Tyr35 plays a particularly important role in defining the conformation of the trypsin-binding site of BPTI.

A new method that utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the hydrogen/deuterium (H/D) exchange properties of proteins was developed for measuring the thermodynamic properties of protein-ligand complexes in solution. Dissociation constants (Kd values) determined by the method for five model protein-ligand complexes that included those with small molecules, nucleic acids, peptides, and other proteins were generally in good agreement with Kd values measured by conventional methods. Important experimental advantages of the described method over existing methods include: the ability to make measurements in a high-throughput and automated fashion, the ability to make measurements using only picomole quantitities of protein, and the ability to analyze either purified or unpurified protein-ligand complexes.

An explosion of in vitro experimental data on the folding of proteins has revealed many examples of folding in the millisecond or faster timescale, often occurring in the absence of stable intermediate states. We review experimental methods for measuring fast protein folding kinetics, and then discuss various analytical models used to interpret these data. Finally, we classify general mechanisms that have been proposed to explain fast protein folding into two catagories, heterogeneous and homogeneous, reflecting the nature of the transition state. One heterogeneous mechanism, the diffusion-collision mechanism, can be used to interpret experimental data for a number of proteins.

The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.

The yeast prolyl isomerase, Ess1, has recently been shown to interact via its WW domain with the hyperphosphorylated form of the RNA polymerase II C-terminal domain (CTD). We have investigated folding of the Ess1 WW domain and its binding to peptides representing the CTD by circular dichroism and fluorescence. Ess1 WW folds and unfolds reversibly, but in the absence of ligand is only marginally stable with a melting temperature of 19 degrees C. The WW domain is stabilized by the addition of anionic ligands, namely, chloride, inorganic phosphate, phosphoserine, and phosphorylated CTD peptides. Dissociation constants were measured to be 70--100 microM for CTD peptides phosphorylated at one serine, and 16--21 microM for peptides with two or more phosphorylated serines. Weaker or no affinity was observed for nonphosphorylated CTD peptides. There is surprisingly little difference in the affinity for peptides phosphorylated at Ser 2 or Ser 5 of the consensus repeat, or for peptides with different patterns of multiple phosphorylation. The binding of Ess1 to phosphorylated CTD peptides is consistent with a model wherein the WW domain positions Ess1 to catalyze isomerization of the many pSer--Pro peptide bonds in the phosphorylated CTD. We suggest that cis/trans isomerization of prolyl peptide bonds plays a crucial role in CTD function during eukaryotic transcription.

The folding and unfolding kinetics of the B-domain of staphylococcal protein A, a small three-helix bundle protein, were probed by NMR. The lineshape of a single histidine resonance was fit as a function of denaturant to give folding and unfolding rate constants. The B-domain folds extremely rapidly in a two-state manner, with a folding rate constant of 120,000 s-1, making it one of the fastest-folding proteins known. Diffusion-collision theory predicts folding and unfolding rate constants that are in good agreement with the experimental values. The apparent rate constant as a function of denaturant ('chevron plot') is predicted within an order of magnitude. Our results are consistent with a model whereby fast-folding proteins utilize a diffusion-collision mechanism, with the preorganization of one or more elements of secondary structure in the unfolded protein.

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 alpha/beta 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 (K(fold)) at 37 degrees C for the P protein was determined to be 0.0071 +/- 0.0005 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 (K(a)'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.

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric lambda repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.

The folding of coiled coil peptides has traditionally been interpreted in terms of native dimer and unfolded monomers. Calculations using AGADIR and experimental studies of fragments suggest that the monomers of the coiled coil peptide, GCN4-p1, contain significant residual helical structure. A simple model based on diffusion-collision theory predicts not only the measured folding rate within an order of magnitude, but also predicts remarkably well the effect of alanine to glyXcine mutations. We suggest that intrinsic helix stability is a major determinant of the folding rate of the GCN4 coiled coil., (Copyright 1999 Academic Press.)

A hydrogen bond between the buried residues Asp 14 and Ser 77 in monomeric lambda repressor has been removed by mutation of these residues to alanine. Double mutant cycles show that the interaction stabilizes the native state of the protein by 1.5 kcal/mol. Removal of the interaction affects mainly the unfolding rates and not the folding rates, suggesting that this hydrogen bond is not substantially formed in the rate-limiting steps in the folding pathways of the protein. Mutations in two versions of lambda6-85, wild type and the faster folding G46A/G48A (WT), show similar effects. Diffusion-collision correctly predicts the behavior of WT but not of wild type. Our analysis suggests that folding of helix 3 is a crucial slow step along the various folding pathways and generally occurs before the formation of the 14-77 hydrogen bond. Experiments removing tertiary interactions, combined with experiments altering helical stability and diffusion-collision calculations, provide a strategy to unravel the folding mechanisms of small helical proteins.

15Nitrogen relaxation experiments were used to characterize the backbone dynamics of two modified forms of bovine pancreatic trypsin inhibitor (BPTI). In one form, the disulfide between Cys14 and Cys38 in the wild-type protein was selectively reduced and methylated to generate an analog of the final intermediate in the disulfide-coupled folding pathway. The second form was generated by similarly modifying a mutant protein in which Tyr35 was replaced with Gly (Y35G). For both selectively reduced proteins, the overall conformation of native BPTI was retained, and the relaxation data for these proteins were compared with those obtained previously with the native wild-type and Y35G proteins. Removing the disulfide from either protein had only small effects on the observed longitudinal relaxation rates (R1) or heteronuclear cross relaxation rates (nuclear Overhauser effect), suggesting that the 14-38 disulfide has little influence on the fast (ps to ns) backbone dynamics of either protein. In the wild-type protein, the pattern of residues undergoing slower (micros to ms) internal motions, reflected in unusually large transverse relaxation rates (R2), was also largely unaffected by the removal of this disulfide. It thus appears that the large R2 rates previously observed in native wild-type protein are not a direct consequence of isomerization of the 14-38 disulfide. In contrast with the wild-type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amides displaying large R2 rates. In addition, the frequencies of the backbone motions in the modified protein, estimated from R2 values measured at multiple refocusing delays, appear to span a wider range than those seen in native Y35G BPTI. Together, these observations suggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14-38 disulfide and that formation of this bond may lead to a substantial loss of conformational entropy. These effects may account for the previous observation that the Y35G substitution greatly destabilizes the disulfide. The results also demonstrate that the disulfide and the buried side-chain influence the dynamics of the folded protein in a highly cooperative fashion, with the effects of removing either being much greater in the absence of the other., (Copyright 1998 Academic Press)

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids "sense" the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins (lambda repressor fragment 6-85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.

A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.

The development of a quantitative kinetic scheme is a central goal in mechanistic studies of biological phenomena. For fast-folding proteins, which lack experimentally observable kinetic intermediates, a quantitative kinetic scheme describing the order and rate of events during folding has yet to be developed. In the present study, the folding mechanism of monomeric lambda repressor is described using the diffusion-collision model and estimates of intrinsic alpha-helix propensities. The model accurately predicts the folding rates of the wild-type protein and five of eight previously studied Ala left and right arrow Gly variants and suggests that the folding mechanism is distributed among multiple pathways that are highly sensitive to the amino acid sequence. For example, the model predicts that the wild-type protein folds through a small number of pathways with a folding time of 260 micros. However, the folding of a variant (G46A/G48A) is predicted to fold through a large number of pathways with a folding time of 12 micros. Both folding times quantitatively agree with the experimental values at 37 degrees C extrapolated to 0 M denaturant. The quantitative nature of the diffusion-collision model allows for rigorous experimental tests of the theory.

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 micros time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.