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With the development of sophisticated pulsed field gradient- and phase cycling-approaches for suppressing certain coherence transfer pathways and selecting for others it is sometimes easy to forget that the process is not flawless. In some cases artifacts can emerge because unwanted transfers are immune to the phase cycle or the application of gradients. We consider here a simple 1 H, 13 C HMQC pulse scheme and show that imperfections in the single 1 H 180° refocusing pulse can give rise to small artifacts in methyl spectra that cannot be eliminated through extensive phase cycling or the use of gradients, but that are easily removed when the pulse is of the composite variety.

Biological molecules are often highly dynamic, and this flexibility can be critical for function. The large range of sampled timescales and the fact that many of the conformers that are continually explored are only transiently formed and sparsely populated challenge current biophysical approaches. Solution nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for characterizing biomolecular dynamics in detail, even in cases where excursions involve short-lived states. Here, we briefly review a number of NMR experiments for studies of biomolecular dynamics on the microsecond-to-second timescale and focus on applications to protein and nucleic acid systems that clearly illustrate the functional relevance of motion in both health and disease.

The 20S proteasome core particle (20S CP) plays an integral role in cellular homeostasis by degrading proteins no longer required for function. The process is, in part, controlled via gating residues localized to the ends of the heptameric barrel-like CP structure that occlude substrate entry pores, preventing unregulated degradation of substrates that might otherwise enter the proteasome. Previously, we showed that the N-terminal residues of the α-subunits of the CP from the archaeon Thermoplasma acidophilum are arranged such that, on average, two of the seven termini are localized inside the lumen of the proteasome, thereby plugging the entry pore and functioning as a gate. However, the mechanism of gating remains unclear. Using solution NMR and a labeling procedure in which a series of mixed proteasome rings are prepared such that the percentage of gate-containing subunits is varied, we address the energetics of gating and establish whether gating is a cooperative process involving the concerted action of residues from more than a single protomer. Our results establish that the intrinsic probability of a gate entering the lumen favors the in state by close to 20-fold, that entry of each gate is noncooperative, with the number of gates that can be accommodated inside the lumen a function of the substrate entry pore size and the bulkiness of the gating residues. Insight into the origin of the high affinity for the in state is obtained from spin-relaxation experiments. More generally, our approach provides an avenue for dissecting interactions of individual protomers in homo-oligomeric complexes., Competing Interests: The authors declare no conflict of interest.

Transient excursions of native protein states to functionally relevant higher energy conformations often occur on the μs-ms timescale. NMR spectroscopy has emerged as an important tool to probe such processes using techniques such as Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and Chemical Exchange Saturation Transfer (CEST). The extraction of kinetic and structural parameters from these measurements is predicated upon mathematical modeling of the resulting relaxation profiles, which in turn relies on knowledge of the initial magnetization conditions at the start of the CPMG/CEST relaxation elements in these experiments. Most fitting programs simply assume initial magnetization conditions that are given by equilibrium populations, which may be incorrect in certain implementations of experiments. In this study we have quantified the systematic errors in extracted parameters that are generated from analyses of CPMG and CEST experiments using incorrect initial boundary conditions. We find that the errors in exchange rates (k ex ) and populations (p E ) are typically small (<10 %) and thus can be safely ignored in most cases. However, errors become larger and cannot be fully neglected (20-40 %) as k ex falls near the lower limit of each method or when short CPMG/CEST relaxation elements are used in these experiments. The source of the errors can be rationalized and their magnitude given by a simple functional form. Despite the fact that errors tend to be small, it is recommended that the correct boundary conditions be implemented in fitting programs so as to obtain as robust estimates of exchange parameters as possible.

In the past several decades solution NMR spectroscopy has emerged as a powerful technique for the study of the structure and dynamics of proteins, providing detailed insights into biomolecular function. Herein, I provide a summary of two important areas of application, focusing on NMR studies of (i) supramolecular systems with aggregate molecular masses in the hundreds of kilodaltons and of (ii) sparsely populated and transiently formed protein states that are thermally accessible from populated ground-state conformers. The critical role of molecular dynamics in function is emphasized, highlighting the utility of the NMR technique in providing such often elusive information., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

Sparsely populated and transiently formed protein conformers can play key roles in many biochemical processes. Understanding the structure function paradigm requires, therefore, an atomic-resolution description of these rare states. However, they are difficult to study because they cannot be observed using standard biophysical techniques. In the past decade, NMR methods have been developed for structural studies of these elusive conformers, focusing primarily on backbone (1)H, (15)N and (13)C nuclei. Here we extend the methodology to include side chains by developing a (13)C-based chemical exchange saturation transfer experiment for the assignment of side-chain aliphatic (13)C chemical shifts in uniformly (13)C labeled proteins. A pair of applications is provided, involving the folding of β-sheet Fyn SH3 and α-helical FF domains. Over 96% and 89% of the side-chain (13)C chemical shifts for excited states corresponding to the unfolded conformation of the Fyn SH3 domain and a folding intermediate of the FF domain, respectively, have been obtained, providing insight into side-chain packing and dynamics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Large macromolecular assemblies, so-called molecular machines, are critical to ensuring proper cellular function. Understanding how proper function is achieved at the atomic level is crucial to advancing multiple avenues of biomedical research. Biophysical studies often include X-ray diffraction and cryo-electron microscopy, providing detailed structural descriptions of these machines. However, their inherent flexibility has complicated an understanding of the relation between structure and function. Solution NMR spectroscopy is well suited to the study of such dynamic complexes, and continued developments have increased size boundaries; insights into function have been obtained for complexes with masses as large as 1 MDa. We highlight methyl-TROSY (transverse relaxation optimized spectroscopy) NMR, which enables the study of such large systems, and include examples of applications to several cellular machines. We show how this emerging technique contributes to an understanding of cellular function and the role of molecular plasticity in regulating an array of biochemical activities.

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the β positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the γ positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstrated with an application to the four-helix bundle colicin E7 immunity protein, Im7, which folds via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. The extracted (1)H chemical shift differences at side-chain positions provide valuable restraints in structural studies of invisible, excited states, complementing backbone chemical shifts that are available from existing relaxation dispersion experiments.

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby (13)CH(3) methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-(2)H; β-(2)H;γ-(13)C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr (13)C,(1)H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[(13)C,(1)H]-Thr.

A method for quantifying millisecond time scale exchange in proteins is presented based on scaling the rate of chemical exchange using a 2D (15)N, (1)H(N) experiment in which (15)N dwell times are separated by short spin-echo pulse trains. Unlike the popular Carr-Purcell-Meiboom-Gill (CPMG) experiment where the effects of a radio frequency field on measured transverse relaxation rates are quantified, the new approach measures peak positions in spectra that shift as the effective exchange time regime is varied. The utility of the method is established through an analysis of data recorded on an exchanging protein-ligand system for which the exchange parameters have been accurately determined using alternative approaches. Computations establish that a combined analysis of CPMG and peak shift profiles extends the time scale that can be studied to include exchanging systems with highly skewed populations and exchange rates as slow as 20 s(-1).

Biological macromolecules are highly flexible and continually undergo conformational fluctuations on a broad spectrum of timescales. It has long been recognized that dynamics have an important role in the action of these molecules. However, the relationship between molecular function and motion is extremely challenging to delineate, because the conformational space available to macromolecules is vast and the relevant excursions can be infrequent and short-lived. Recent advances in solution nuclear magnetic resonance (NMR) spectroscopy permit biomolecular dynamics to be observed with unprecedented detail. Applications of these new NMR techniques to the study of fundamental processes such as binding and catalysis have provided new insights into how living systems operate at an atomic level.

The three-dimensional structures of macromolecules fluctuate over a wide range of time-scales. Separating the individual dynamic processes according to frequency is of importance in relating protein motions to biological function and stability. We present here a general NMR method for the specific characterization of microsecond motions at backbone positions in proteins even in the presence of other dynamics such as large-amplitude nanosecond motions and millisecond chemical exchange processes. The method is based on measurement of relaxation rates of four bilinear coherences and relies on the ability of strong continuous radio frequency fields to quench millisecond chemical exchange. The utility of the methodology is demonstrated and validated through two specific examples focusing on the thermo-stable proteins, ubiquitin and protein L, where it is found that small-amplitude microsecond dynamics are more pervasive than previously thought. Specifically, these motions are localized to alpha helices, loop regions, and regions along the rim of beta sheets in both of the proteins examined. A third example focuses on a 28 kDa ternary complex of the chaperone Chz1 and the histones H2A.Z/H2B, where it is established that pervasive microsecond motions are localized to a region of the chaperone that is important for stabilizing the complex. It is further shown that these motions can be well separated from extensive millisecond dynamics that are also present and that derive from exchange of Chz1 between bound and free states. The methodology is straightforward to implement, and data recorded at only a single static magnetic field are required.

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.

A labeling scheme is introduced that facilitates the measurement of accurate (13)C(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of (13)C enrichment (30-40%) at C(beta) side-chain carbon positions for 15 of the amino acids with little (13)C label at positions one bond removed (approximately 5%). A pair of samples are produced using [1-(13)C]-glucose/NaH(12)CO(3) or [2-(13)C]-glucose as carbon sources with isolated and enriched (>30%) (13)C(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of (13)C(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.

The NMR structure of budding yeast chaperone Chz1 complexed with histones H2A.Z-H2B has been determined. Chz1 forms a long irregular chain capped by two short alpha-helices, and uses both positively and negatively charged residues to stabilize the histone dimer. A molecular model that docks Chz1 onto the nucleosome has implications for its potential functions.

Relaxation dispersion NMR spectroscopy has become a valuable probe of millisecond dynamic processes in biomolecules that exchange between a ground (observable) state and one or more excited (invisible) conformers, in part because chemical shifts of the excited state(s) can be obtained that provide insight into the conformations that are sampled. Here we present a pair of experiments that provide additional structural information in the form of residual dipolar couplings of the excited state. The new experiments record (1)H spin-state selective (13)CO and (13)C(alpha) dispersion profiles under conditions of partial alignment in a magnetic field from which two-bond (1)HN-(13)CO and one-bond (1)H(alpha)-(13)C(alpha) residual dipolar couplings of the invisible conformer can be extracted. These new dipolar couplings complement orientational restraints that are provided through measurement of (1)HN-(15)N residual dipolar couplings and changes in (13)CO chemical shifts upon alignment that have been measured previously for the excited-state since the interactions probed here are not collinear with those previously investigated. An application to a protein-ligand binding reaction is presented, and the accuracies of the extracted excited-state dipolar couplings are established. A combination of residual dipolar couplings and chemical shifts as measured by relaxation dispersion will facilitate a quantitative description of excited protein states.

Currently the main focus of structural biology is the determination of static three-dimensional representations of biomolecules that for the most part correspond to low energy (ground state) conformations. However, it is becoming increasingly well recognized that higher energy structures often play important roles in function as well. Because these conformers are populated to only low levels and are often only transiently formed their study is not amenable to many of the tools of structural biology. In this perspective we discuss the role of CPMG-based relaxation dispersion NMR spectroscopy in characterizing these low populated, invisible states. It is shown that robust methods for measuring both backbone chemical shifts and residual anisotropic interactions in the excited state are in place and that these data provide valuable restraints for structural studies of invisible conformers.

A general method for obtaining quantitative structural information on invisible, excited protein states by solution-based NMR spectroscopy is presented. The approach exploits relaxation dispersion techniques in which changes in chemical shifts between ground and excited states are monitored in solutions with and without small amounts of residual molecular alignment. This allows the calculation of differences in chemical shifts induced by alignment that can be directly related to molecular structure, in cases where the orientation and magnitude of the chemical-shift tensor are well defined. An example using carbonyl chemical shifts as probes of a protein-ligand binding reaction is presented to illustrate and validate the method.

Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy has evolved into a powerful approach for the study of low populated, invisible conformations of biological molecules. One of the powerful features of the experiment is that chemical shift differences between the exchanging conformers can be obtained, providing structural information about invisible excited states. Through the development of new labeling approaches and NMR experiments it is now possible to measure backbone 13C(alpha) and 13CO relaxation dispersion profiles in proteins without complications from 13C-13C couplings. Such measurements are presented here, along with those that probe exchange using 15N and 1HN nuclei. A key experimental design has been the choice of an exchanging system where excited-state chemical shifts were known from independent measurement. Thus it is possible to evaluate quantitatively the accuracy of chemical shift differences obtained in dispersion experiments and to establish that in general very accurate values can be obtained. The experimental work is supplemented by computations that suggest that similarly accurate shifts can be measured in many cases for systems with exchange rates and populations that fall within the range of those that can be quantified by relaxation dispersion. The accuracy of the extracted chemical shifts opens up the possibility of obtaining quantitative structural information of invisible states of the sort that is now available from chemical shifts recorded on ground states of proteins.

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.

A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.

Single-molecule fluorescence experiments have shown that the conformation of the complex between Escherichia coli general NAD(P)H:flavin oxidoreductase (FRE) and flavin adenine dinucleotide (FAD) fluctuates over a range of timescales between 10(-4) and 1 s. Here we use (15)N and (13)C relaxation dispersion NMR methods to study millisecond-timescale dynamics in the complex. In this time regime, the protein is extremely flexible, with residues that undergo conformational exchange located throughout the molecule. Three distinct regions of dynamics are quantified, with two of them involving residues making contact to the donor (Tyr-35) and acceptor (FAD) sites that participate in the electron transfer reaction monitored in single-molecule experiments. Modulation of the donor-acceptor distance through these conformational exchange processes, occurring with rates of approximately 400 and 1,200 s(-1) (22 degrees C), affects the rate of electron transfer and partially accounts for the range of the observed dynamics monitored in the fluorescence experiments.

A new labeling strategy is presented that greatly facilitates the measurement of 2H spin relaxation rates in RNA molecules as a probe of pico- to nanosecond time scale dynamics. In this labeling scheme the sugar positions are uniformly 13C-labeled, with position 2' protonated and all other sites on the sugar deuterated. Pulse sequences are presented for measurement of 2H R1 and R2 relaxation rates at positions 1', 3', and 4' with sensitivity gains that are on the order of 5-fold relative to previous methods that employed random fractional deuteration. The improved sensitivity is transformative and facilitates the study of motion in moderately sized RNA molecules with good sensitivity. The utility of the approach is demonstrated with an application to HIV-2 TAR, where the site-specific measures of molecular dynamics at sugar positions obtained here complement previous studies of dynamics at aromatic sites in the molecule.

Protein folding kinetic data have been obtained for the marginally stable N-terminal Src homology 3 domain of the Drosophila protein drk (drkN SH3) in an investigation of the hydrodynamic properties of its folding transition state. Due to the presence of NMR resonances of both folded and unfolded states at equilibrium, kinetic data can be derived from NMR magnetization transfer techniques under equilibrium conditions. Kinetic analysis as a function of urea (less than approximately 1 M) and glycerol enables determination of alpha values, measures of the energetic sensitivity of the transition state to the perturbation relative to the end states of the protein folding reaction (the folded and unfolded states). Both end states have previously been studied experimentally by NMR spectroscopic and other biophysical methods in great detail and under nondenaturing conditions. Combining these results with the kinetic folding data obtained here, we can characterize the folding transition state without requiring empirical models for the unfolded state structure. We are thus able to give a reliable measure of the solvent-accessible surface area of the transition state of the drkN SH3 domain (4730 +/- 360 A(2)) based on urea titration data. Glycerol titration data give similar results and additionally demonstrate that folding of this SH3 domain is dependent on solvent viscosity, which is indicative of at least partial hydration of the transition state. Because SH3 domains appear to fold by a common folding mechanism, the data presented here provide valuable insight into the transition states of the drkN and other SH3 domains.

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.

The development of isotope labeling methodology has had a significant impact on NMR studies of high-molecular-weight proteins and macromolecular complexes. Here we review some of this methodology that has been developed and used in our laboratory. In particular, experimental protocols are described for the production of highly deuterated, uniformly 15N- and 13C-labeled samples of large proteins, with optional incorporation of selective isotope labels into methyl groups of isoleucine, leucine and valine residues. Various types of methyl labeling schemes are assessed, and the utility of different methyl labeling strategies is highlighted for studies ranging from protein structure determination to the investigation of side-chain dynamics. In the case of malate synthase G (MSG), the time frame of the whole preparation, including the protein refolding step, is about 70 h.

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.

Solution NMR spectroscopy is rapidly becoming an important technique for the study of membrane protein structure and dynamics. NMR experiments on large perdeuterated proteins typically exploit the favorable relaxation properties of backbone amide (15)N-(1)H groups to obtain sequence-specific chemical shift assignments, structural restraints, and a wide range of dynamics information. These methods have proven successful in the study of the outer membrane enzyme, PagP, not only for obtaining the global fold of the protein but also for characterizing in detail the conformational fluctuations that are critical to its activity. NMR methods can also be extended to take advantage of slowly relaxing methyl groups, providing additional probes of structure and dynamics at side chain positions. The current work on PagP demonstrates how solution NMR can provide a unique atomic resolution description of the dynamic processes that are key to the function of many membrane protein systems.

Relaxation in methyl groups is strongly influenced by cross-correlated interactions involving the methyl dipoles. One of the major interference effects results from intra-methyl (1)H-(13)C, (1)H-(1)H dipolar interactions, leading to significant differences in the relaxation of certain multiplet components that contribute to double- and zero-quantum (1)H-(13)C spectra. NMR experiments are presented for the measurement of this differential relaxation effect. It is shown that this difference in relaxation between double- and zero-quantum multiplet components can be used as a sensitive reporter of side chain dynamics and that accurate methyl axis order parameters can be measured in proteins that tumble with correlation times greater than approximately 5 ns.

New relaxation dispersion experiments are presented that probe millisecond time-scale dynamical processes in proteins. The experiments measure the relaxation of (1)H-(15)N multiple-quantum coherence as a function of the rate of application of either (1)H or (15)N refocusing pulses during a constant time relaxation interval. In contrast to the dispersion profiles generated from more conventional (15)N((1)H) single-quantum relaxation experiments that depend on changes in (15)N((1)H) chemical shifts between exchanging states, (1)H-(15)N multiple-quantum dispersions are sensitive to changes in the chemical environments of both (1)H and (15)N spins. The resulting multiple-quantum relaxation dispersion profiles can, therefore, be quite different from those generated by single-quantum experiments, so that an analysis of both single- and multiple-quantum profiles together provides a powerful approach for obtaining robust measures of exchange parameters. This is particularly the case in applications to protonated proteins where other methods for studying exchange involving amide proton spins are negatively influenced by contributions from neighboring protons. The methodology is demonstrated on protonated and perdeuterated samples of a G48M mutant of the Fyn SH3 domain that exchanges between folded and unfolded states in solution.

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on (15)N, natural abundance (13)C samples. The experiment exploits the fact that transfers of magnetization from (15)N to (13)CO and from (15)N to (13)C(alpha) (and back) proceed independently for samples that are not enriched in (13)C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM (15)N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz.

Recently developed 2H spin relaxation experiments are applied to study the dynamics of methyl-containing side-chains in the B1 domain of protein L and in a pair of point mutants of the domain, F22L and A20V. X-ray and NMR studies of the three variants of protein L studied here establish that their structures are very similar, despite the fact that the F22L mutant is 3.2kcal/mol less stable. Measurements of methyl 2H spin relaxation rates, which probe dynamics on a picosecond-nanosecond time scale, and three-bond 3J(Cgamma-CO), 3J(Cgamma-N) and 3J(Calpha-Cdelta) scalar coupling constants, which are sensitive to motion spanning a wide range of time-scales, reveal changes in the magnitude of side-chain dynamics in response to mutation. Observed differences in the time-scale of motions between the variants have been related to changes in energetic barriers. Of interest, several of the residues with different motional properties across the variants are far from the site of mutation, suggesting the presence of long-range interactions within the protein that can be probed through studies of dynamics.

An (15)N off-resonance R(1rho) spin relaxation study of an L99A point mutant of T4 lysozyme is presented. Previous CPMG-based relaxation dispersion studies of exchange in this protein have established that the molecule interconverts between a populated ground state and an excited state (3.4%) with an exchange rate constant of 1450 s(-1) at 25 degrees C. It is shown that for the majority of residues in this protein the offset dependence of the R(1rho) relaxation rates cannot be well fit using models which are only valid in the fast exchange regime. In contrast, a recently derived expression by Trott and Palmer (J. Magn. Reson., 154, 157-160, 2002) which is valid over a wider window of exchange than other relations, is shown to fit the data well. Values of (signed) chemical shift differences between exchanging sites have been extracted and are in reasonable agreement with shift differences measured using CPMG methods. A set of simulations is presented which help establish the exchange regimes that are best suited to analysis by off-resonance R(1rho) techniques.

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.)