Search

The determination of a protein's structure is often a first step towards the development of a mechanistic understanding of its function. Considerable advances in computational protein structure prediction have been made in recent years, with AlphaFold2 (AF2) emerging as the primary tool used by researchers for this purpose. While AF2 generally predicts accurate structures of folded proteins, we present here a case where AF2 incorrectly predicts the structure of a small, folded and compact protein with high confidence. This protein, pro-interleukin-18 (pro-IL-18), is the precursor of the cytokine IL-18. Interestingly, the structure of pro-IL-18 predicted by AF2 matches that of the mature cytokine, and not the corresponding experimentally determined structure of the pro-form of the protein. Thus, while computational structure prediction holds immense promise for addressing problems in protein biophysics, there is still a need for experimental structure determination, even in the context of small well-folded, globular proteins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [H.W. is a co-founder and chair of the scientific advisory board of Ventus Therapeutics. This relationship did not influence this study]., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments., Competing Interests: The authors declare no conflict of interest.

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD1 2SH ), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD1 2SH ; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer., Competing Interests: The authors declare no conflict of interest.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD1(2SH)). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD1(2SH) has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization.

Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18-R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.

Understanding the mechanisms by which proteins fold into their three-dimensional structures, including a description of the intermediates that are formed during the folding process, remains a goal of protein science. Most studies are performed under carefully controlled conditions in which the folding reaction is monitored in a buffer solution that is far from the natural milieu of the cell. Here, we have used (13)C and (1)H relaxation dispersion NMR spectroscopy to study folding of the FF domain in both Escherichia coli and Saccharomyces cerevisiae cellular lysates. We find that a conformationally excited state is populated in both lysates, which is very similar in structure to a folding intermediate observed in previous studies in buffer, with the kinetics and thermodynamics of the interconversion between native and intermediate conformers somewhat changed. The results point to the importance of extending folding studies beyond the test tube yet emphasize that insights can be obtained through careful experiments recorded in controlled buffer solutions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The importance of dynamics to biomolecular function is becoming increasingly clear. A description of the structure-function relationship must, therefore, include the role of motion, requiring a shift in paradigm from focus on a single static 3D picture to one where a given biomolecule is considered in terms of an ensemble of interconverting conformers, each with potentially diverse activities. In this Perspective, we describe how recent developments in solution NMR spectroscopy facilitate atomic resolution studies of sparsely populated, transiently formed biomolecular conformations that exchange with the native state. Examples of how this methodology is applied to protein folding and misfolding, ligand binding, and molecular recognition are provided as a means of illustrating both the power of the new techniques and the significant roles that conformationally excited protein states play in biology.

Friction plays a critical role in protein folding. Frictional forces originating from random solvent and protein fluctuations both retard motion along the folding pathway and activate protein molecules to cross free energy barriers. Studies of friction thus may provide insights into the driving forces underlying protein conformational dynamics. However, the molecular origin of friction in protein folding remains poorly understood because, with the exception of the native conformer, there generally is little detailed structural information on the other states participating in the folding process. Here, we study the folding of the four-helix bundle FF domain that proceeds via a transiently formed, sparsely populated compact on-pathway folding intermediate whose structure was elucidated previously. Because the intermediate is stabilized by both native and nonnative interactions, friction in the folding transition between intermediate and folded states is expected to arise from intrachain reorganization in the protein. However, the viscosity dependencies of rates of folding from or unfolding to the intermediate, as established by relaxation dispersion NMR spectroscopy, clearly indicate that contributions from internal friction are small relative to those from solvent, so solvent frictional forces drive the folding process. Our results emphasize the importance of solvent dynamics in mediating the interconversion between protein configurations, even those that are highly compact, and in equilibrium folding/unfolding fluctuations in general.

Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.

A 2D (13)C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded ('ground') state are present in spectra. Fits of (15)N and methyl (13)C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl (13)C transverse relaxation rates for some of the residues of the unfolded ('invisible, excited') state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the (13)C CEST data.

Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal β strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.

Several all-helical single-domain proteins have been shown to fold rapidly (microsecond time scale) to a compact intermediate state and subsequently rearrange more slowly to the native conformation. An understanding of this process has been hindered by difficulties in experimental studies of intermediates in cases where they are both low-populated and only transiently formed. One such example is provided by the on-pathway folding intermediate of the small four-helix bundle FF domain from HYPA/FBP11 that is populated at several percent with a millisecond lifetime at room temperature. Here we have studied the L24A mutant that has been shown previously to form nonnative interactions in the folding transition state. A suite of Carr-Purcell-Meiboom-Gill relaxation dispersion NMR experiments have been used to measure backbone chemical shifts and amide bond vector orientations of the invisible folding intermediate that form the input restraints in calculations of atomic resolution models of its structure. Despite the fact that the intermediate structure has many features that are similar to that of the native state, a set of nonnative contacts is observed that is even more extensive than noted previously for the wild-type (WT) folding intermediate. Such nonnative interactions, which must be broken prior to adoption of the native conformation, explain why the transition from the intermediate state to the native conformer (millisecond time scale) is significantly slower than from the unfolded ensemble to the intermediate and why the L24A mutant folds more slowly than the WT.

Proteins can sample conformational states that are critical for function but are seldom detected directly because of their low occupancies and short lifetimes. In this work, we used chemical shifts and bond-vector orientation constraints obtained from nuclear magnetic resonance relaxation dispersion spectroscopy, in concert with a chemical shift-based method for structure elucidation, to determine an atomic-resolution structure of an "invisible" folding intermediate of a small protein module: the FF domain. The structure reveals non-native elements preventing formation of the native conformation in the carboxyl-terminal part of the protein. This is consistent with the kinetics of folding in which a well-structured intermediate forms rapidly and then rearranges slowly to the native state. The approach introduces a general strategy for structure determination of low-populated and transiently formed protein states.

Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a "high-resolution" picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.

Biological function depends on molecular dynamics that lead to excursions from highly populated ground states to much less populated excited states. The low populations and the transient formation of such excited states render them invisible to the conventional methods of structural biology. Thus, while detailed pictures of ground-state structures of biomolecules have emerged over the years, largely through X-ray diffraction and solution nuclear magnetic resonance (NMR) spectroscopy studies, much less structural data has been accumulated on the conformational properties of the invisible excited states that are necessary to fully explain function. NMR spectroscopy is a powerful tool for studying conformational dynamics because it is sensitive to dynamics over a wide range of time scales, extending from picoseconds to seconds and because information is, in principle, available at nearly every position in the molecule. Here an NMR method for quantifying millisecond time scale dynamics that involve transitions between different molecular conformations is described. The basic experimental approach, termed relaxation dispersion NMR spectroscopy, is outlined to provide the reader with an intuitive feel for the technology. A variety of different experiments that probe conformational exchange at different sites in proteins are described, including a brief summary of data-fitting procedures to extract both the kinetic and thermodynamic properties of the exchange process and the structural features of the invisible excited states along the exchange pathway. It is shown that the methodology facilitates detection of intermediates and other excited states that are populated at low levels, 0.5% or higher, that cannot be observed directly in spectra, so long as they exchange with the observable ground state of the protein on the millisecond time scale. The power of the methodology is illustrated by a detailed application to the study of protein folding of the small modular SH3 domain. The kinetics and thermodynamics that describe the folding of this domain have been characterized through the effects of temperature, pressure, side-chain deuteration, and mutation, and the structural features of a low-populated folding intermediate have been assessed. Despite the fact that many previous studies have shown that SH3 domains fold via a two-state mechanism, the NMR methods presented unequivocally establish the presence of an on-pathway folding intermediate. The unique capabilities of NMR relaxation dispersion follow from the fact that large numbers of residues can be probed individually in a single experiment. By contrast, many other forms of spectroscopy monitor properties that are averaged over all residues in the molecule or that make use of only one or two reporters. The NMR methodology is not limited to protein folding, and applications to enzymatic catalysis, binding, and molecular recognition are beginning to emerge.

Experimental studies of protein folding frequently are consistent with two-state folding kinetics. However, recent NMR relaxation dispersion studies of several fast-folding mutants of the Fyn Src homology 3 (SH3) domain have established that folding proceeds through a low-populated on-pathway intermediate, which could not be detected with stopped-flow experiments. The dispersion experiments provide precise kinetic and thermodynamic parameters that describe the folding pathway, along with a detailed site-specific structural characterization of both the intermediate and unfolded states from the NMR chemical shifts that are extracted. Here we describe NMR relaxation dispersion Phi-value analysis of the A39V/N53P/V55L Fyn SH3 domain, where the effects of suitable point mutations on the energy landscape are quantified, providing additional insight into the structure of the folding intermediate along with per-residue structural information of both rate-limiting transition states that was not available from previous studies. In addition to the advantage of delineating the full three-state folding pathway, the use of NMR relaxation dispersion as opposed to stopped-flow kinetics to quantify Phi values facilitates their interpretation because the obtained chemical shifts monitor any potential structural changes along the folding pathway that might be introduced by mutation, a significant concern in their analysis. Phi-Value analysis of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the beta(3)-beta(4)-hairpin already is formed in the first transition state, whereas strand beta(1), which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts.

A series of experiments are presented that provide an exchange-free measure of dipole-dipole (15)N transverse relaxation, R(dd), that can then be substituted for (15)N R(1rho) or R(2) rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving (1)H-(15)N longitudinal order, anti-phase (1)H and (15)N single-quantum coherences, and (1)H-(15)N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that R(dd) values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1-2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b(562) that shows large contributions to (15)N R(1rho) relaxation from chemical exchange.

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.

A relaxation dispersion pulse scheme is presented for quantifying chemical exchange processes in proteins that exploits 1H chemical shifts as probes of changes in conformation. The experiment selects 1H single-quantum magnetization from the I = 1/2 manifolds of the methyl group, which behave like AX spin systems, while suppressing coherences that derive from the 3/2 manifold that are extremely sensitive to pulse imperfections and that would otherwise severely compromise the accuracy of the experiment. The utility of the sequence is first demonstrated with an application to a protein system that is known not to undergo chemical exchange and flat dispersion profiles are obtained. Subsequently, the methodology is applied to study the folding of a G48M mutant of the Fyn SH3 domain that has been shown previously to undergo exchange between folded and unfolded states on the millisecond time scale.

Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data.

Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F <--> I <--> U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding.

The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales.

There is growing evidence that structural flexibility plays a central role in the function of protein molecules. Many of the experimental data come from nuclear magnetic resonance (NMR) spectroscopy, a technique that allows internal motions to be probed with exquisite time and spatial resolution. Recent methodological advancements in NMR have extended our ability to characterize protein dynamics and promise to shed new light on the mechanisms by which these molecules function. Here, we present a brief overview of some of the new methods, together with applications that illustrate the level of detail at which protein motions can now be observed.

A major challenge to the study of protein folding is the fact that intermediate states along the reaction pathway are generally unstable and thus difficult to observe. Recently developed NMR relaxation dispersion experiments present an avenue to accessing such states, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to approximately 1%) populations at equilibrium. We have employed these techniques to study the three-state folding reaction of the G48M Fyn SH3 domain. Using (13)C-, (1)H-, and (15)N-based methods, we have characterized backbone and side-chain interactions in the folded, unfolded, intermediate, and transition states, thereby mapping the energy landscape of the protein. We find that the intermediate, populated to approximately 1%, contains nativelike structure in a central beta-sheet, and is disordered at the amino and carboxy termini. The intermediate is stabilized by side-chain van der Waals contacts, yet (13)C chemical shifts indicate that methyl-containing residues remain disordered. This state has a partially structured backbone and a collapsed yet mobile hydrophobic core and thus closely resembles a molten globule. Nonpolar side-chain contacts are formed in the unfolded-intermediate transition state; these interactions are disrupted in the intermediate-folded transition state, possibly allowing side chains to rearrange as they adopt the native packing configuration. This work illustrates the power of novel relaxation dispersion experiments in characterizing excited states that are "invisible" in even the most sensitive of NMR experiments.

The three-site exchange folding reaction of an (15)N-labeled, highly deuterated Gly48Met mutant of the Fyn SH3 domain has been characterized at 25 degrees C using a suite of six CPMG-type relaxation dispersion experiments that measure exchange contributions to backbone (1)H and (15)N transverse relaxation rates in proteins. It is shown that this suite of experiments allows the extraction of all the parameters of this multisite exchange process in a robust manner, including chemical shift differences between exchanging states, from a data set recorded at only a single temperature. The populations of the exchanging folded, intermediate, and unfolded states that are fit are 94, 0.7, and 5%, respectively. Despite the small fraction of the intermediate, structural information is obtained for this state that is consistent with the picture of SH3 domain folding that has emerged from other studies. Taken together, the six dispersion experiments facilitate the complete reconstruction of (1)H-(15)N correlation spectra for the unfolded and intermediate states that are "invisible" in even the most sensitive of NMR experiments.

Protein folding kinetic data have been obtained for the marginally stable N-terminal SH3 domain of the Drosophila protein drk as a function of pH in order to investigate the electrostatic properties of Asp8 in the folding transition state ensemble. The slow exchange between folded and unfolded forms of the protein gives rise to separate NMR resonances for both folded and unfolded states at equilibrium. As a result, kinetic data can be derived from magnetization transfer between these two states without the need for denaturants. Using the fact that ionization of Asp8 dominates the electrostatic behavior of the protein between pH 2 and 3, along with pKa values for titrating groups in both folded and unfolded states that have been determined in a previous study, values of 2.9 +/- 0.1 and 3.3 +/- 0.2 are obtained for the pKa of Asp8 in the transition state for the wild-type protein and for a His7Ala mutant, respectively. The data are consistent with the partial formation in the transition state ensemble of an Asp8 side chain carboxylate-a Lys21 backbone amide interaction that represents a highly conserved contact in folded SH3 domains.

A quantitative analysis of 2D (1)H-(15)N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by recording 2D projection planes from HNCO data sets. Applications are presented involving the measurement of (15)N T(1rho) relaxation rates in a high molecular weight protein, malate synthase G, and in a system that exchanges between folded and unfolded states, the drkN SH3 domain. By supplementing relaxation data recorded in the conventional way as a series of 2D (1)H-(15)N data sets with a series of a pair of projection planes the number of dynamics probes is increased significantly for both systems studied.

Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.

Through a mutagenic investigation of Gly-48, a highly conserved position in the Src homology 3 domain, we have discovered a series of amino acid substitutions that are highly destabilizing, yet dramatically accelerate protein folding, some up to 10-fold compared with the wild-type rate. The unique folding properties of these mutants allowed for accurate measurement of their folding and unfolding rates in water with no denaturant by using an NMR spin relaxation dispersion technique. A strong correlation was found between beta-sheet propensity and the folding rates of the Gly-48 mutants, even though Gly-48 lies in an unusual non-beta-strand backbone conformation in the native state. This finding indicates that the accelerated folding rates of the Gly-48 mutants are the result of stabilization of a nonnative beta-strand conformation in the transition-state structure at this position, thus providing the first, to our knowledge, experimentally elucidated example of a mechanism by which folding can occur fastest through a nonnative conformation. We also demonstrate that residues that are most stabilizing in the transition-state structure are most destabilizing in the native state, and also cause the greatest reductions in in vitro functional activity. These data indicate that the unusual native conformation of the Gly-48 position is important for function, and that evolutionary selection for function can result in a domain that folds at a rate far below the maximum possible.

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U(exch), which exists in equilibrium with the folded state, F(exch), under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30-40% larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U(exch) state with those in a denatured state obtained by addition of 2 M guanidinium chloride., (Copyright 2002 Elsevier Science Ltd.)

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ~5 to 300 s-1 involving sparse states with populations as low as ~1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ~100 to ~15,000 µs. A similar folding scheme is established for the wildtype domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES. [ABSTRACT FROM AUTHOR]

• Sparsely populated protein states can play significant biological roles. • NMR spectroscopy can structurally and dynamically characterize 'invisible' protein states. • We review the unique ability of NMR to provide insight into 'invisible' states present in protein folding and misfolding. Proteins interconvert between multiple conformations, including sparsely populated and transiently formed states that are difficult to characterize in structural detail using standard biophysical methods. In some cases, changes to the dynamical equilibria between conformations can lead to pathological protein aggregation and to the disruption of cellular homeostasis. The detection and characterization of lowly populated conformers is therefore crucial for understanding the basis of protein misfolding. NMR spectroscopy is exquisitely sensitive to the conformational dynamics of biomolecules and can be used to study sparsely populated states at the atomic level. Here, we review recent progress toward understanding the roles of sparsely populated, otherwise 'invisible' states present in protein folding and misfolding, where NMR has provided unique insight into folding intermediates, transiently misfolded states, and soluble oligomers that precede amyloid fibril formation. [ABSTRACT FROM AUTHOR]

Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin‐relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG‐based experiment for measuring translational diffusion constants of invisible states using a pulsed‐field gradient approach that exploits methyl 1H triple‐quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state. Diffusion of the invisible state: A CPMG NMR spectroscopy experiment allows measurement of translational diffusion constants of protein excited states. The experiment exploits methyl 1H 3Q coherences and applies encode/decode gradients flanking a CPMG element so that heretofore unmeasurable hydrodynamic properties of invisible states can be obtained. [ABSTRACT FROM AUTHOR]

Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct 15N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained. [ABSTRACT FROM AUTHOR]

Molecular dynamics are essential for protein function. In some cases these dynamics involve the interconversion between ground state, highly populated conformers and less populated higher energy structures ('excited states') that play critical roles in biochemical processes. Here we describe recent advances in NMR spectroscopy methods that enable studies of these otherwise invisible excited states at an atomic level and that help elucidate their important relation to function. We discuss a range of examples from molecular recognition, ligand binding, enzyme catalysis and protein folding that illustrate the role that motion plays in 'funneling' conformers along preferred pathways that facilitate their biological function. [ABSTRACT FROM AUTHOR]