Atomic Resolution Insights into pH Shift Induced Deprotonation Events in LS-Shaped Aß(1-42) Amyloid Fibrils.

Alzheimer's disease is a neurodegenerative disorder associated with the deposition of misfolded aggregates of the amyloid-ß protein (Aß). Aß(1-42) is one of the most aggregation-prone components in senile plaques of AD patients. We demonstrated that relatively homogeneous Aß(1-42) fibrils with one predominant fold visible in solid-state NMR spectra can be obtained at acidic pH. The structure of these fibrils differs remarkably from some other polymorphs obtained at neutral pH. In particular, the entire N-terminal region is part of the rigid fibril core. Here, we investigate the effects of a pH shift on the stability and the fold of these fibrils at higher pH values. Fibril bundling at neutral pH values renders cryo-EM studies impractical, but solid-state NMR spectroscopy, molecular dynamics simulations, and biophysical methods provide residue-specific structural information under these conditions. The LS-fold of the Aß(1-42) fibrils does not change over the complete pH range from pH 2 to pH 7; in particular, the N-terminus remains part of the fibril core. We observe changes in the protonation state of charged residues starting from pH 5 on a residue-specific level. The deprotonation of the C-terminal carboxyl group of A42 in the intermolecular salt bridge with D1 and K28 is slow on the NMR time scale, with a local pKa of 5.4, and local conformations of the involved residues are affected by deprotonation of A42. Thus, we demonstrate that this fibril form is stable at physiological pH values.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Structural details of amyloid ß oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy.

Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid ß (Aß) protein aggregates. Binding of Aß oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer's disease, while an N-terminal soluble fragment of huPrP can sequester Aß oligomers and reduce their toxicity. Synthetic oligomeric Aß species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging. Here, using huPrP to preserve Aß oligomers by coprecipitating them into large heteroassemblies, we investigated the conformations of Aß(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the two C-terminal α-helices. For Aß(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal substantial ß-strand content. Importantly, not all Aß(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aß fibrils suggests that the Aß oligomer preparation represents a heterogeneous mixture of ß-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aß to adopt variable ß-strand-rich conformers. Taken together, our results reveal structural changes in huPrP upon binding to Aß oligomers that suggest a role of the C terminus of huPrP in cell signaling. Trapping Aß(1-42) oligomers by binding to huPrP has proved to be a useful tool for studying the structure of these highly heterogeneous ß-strand-rich assemblies.

A d-enantiomeric peptide interferes with heteroassociation of amyloid-ß oligomers and prion protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of ß-amyloid (Aß) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression. Aß oligomers bind with high affinity to membrane-associated prion protein (PrP), leading to toxic signaling across the cell membrane, which makes the Aß-PrP interaction an attractive therapeutic target. Here, probing this interaction in more detail, we found that both full-length, soluble human (hu) PrP(23-230) and huPrP(23-144), lacking the globular C-terminal domain, bind to Aß oligomers to form large complexes above the megadalton size range. Following purification by sucrose density-gradient ultracentrifugation, the Aß and huPrP contents in these heteroassemblies were quantified by reversed-phase HPLC. The Aß:PrP molar ratio in these assemblies exhibited some limited variation depending on the molar ratio of the initial mixture. Specifically, a molar ratio of about four Aß to one huPrP in the presence of an excess of huPrP(23-230) or huPrP(23-144) suggested that four Aß units are required to form one huPrP-binding site. Of note, an Aß-binding all-d-enantiomeric peptide, RD2D3, competed with huPrP for Aß oligomers and interfered with Aß-PrP heteroassembly in a concentration-dependent manner. Our results highlight the importance of multivalent epitopes on Aß oligomers for Aß-PrP interactions and have yielded an all-d-peptide-based, therapeutically promising agent that competes with PrP for these interactions.

Proton exchange in aqueous urea solutions measured by water-exchange (WEX) NMR spectroscopy and chemical exchange saturation transfer (CEST) imaging in vitro.

PURPOSE: To characterize the proton exchange in aqueous urea solutions using a modified version of the WEX II filter at high magnetic field, and to assess the feasibility of performing quantitative urea CEST MRI on a 3T clinical MR system. METHODS: In order to study the dependence of the exchange-rate constant ksw of urea as a function of pH and T, the WEX-spectra were acquired at 600 MHz from urea solutions in a pH range from 6.4 to 8.0 and a temperature range from T=22∘C to 37∘C . The CEST experiments were performed on a 3T MRI scanner by applying a train of 50 Gaussian-shaped pulses, each 100-millisecond long with a spacing of 100 milliseconds, for saturation. Exchange rates of urea were calculated using the (extended) AREX metric. RESULTS: The results showed that proton exchange in aqueous urea solutions is acid and base catalyzed with the rate constants: ka=(9.95±1.1)×106 l/(mol·s) and kb=(6.21±0.21)×106 l/(mol·s), respectively. Since the urea protons undergo a slow exchange with water protons, the CEST effect of urea can be observed efficiently at 3T. However, in neutral solutions the exchange rate of urea is minimal and cannot be estimated using the quantitative CEST approach. CONCLUSIONS: By means of the WEX-spectroscopy, the kinetic parameters of the proton exchange in urea solutions have been determined. It was also possible to estimate the exchange rates of urea in a broad range of pH values using the CEST method at a clinical scanner.

High-Affinity Binding of Monomeric but Not Oligomeric Amyloid-ß to Ganglioside GM1 Containing Nanodiscs.

The interaction of the amyloid-ß protein (Aß) with neuronal cell membranes plays a crucial role in Alzheimer's disease. Aß undergoes structural changes upon binding to ganglioside GM1 containing membranes leading to altered molecular characteristics of the protein. The physiological role of the Aß interaction with the ganglioside GM1 is still unclear. In order to further elucidate the molecular requirements of Aß membrane binding, we tested different nanodiscs varying in their lipid composition, regarding the charge of the headgroups as well as ganglioside GM1 concentration. Nanodiscs are excellent model membrane systems for studying protein membrane interactions, and we show here their suitability to investigate the membrane interaction of Aß. In particular, we set out to investigate whether the binding activity of GM1 to Aß is specific for the assembly state of Aß and compared the binding affinities of monomeric with oligomeric Aß. Using fluorescence titration experiments, we demonstrate high-affinity binding of Aß(1-40) to GM1 containing nanodiscs, with dissociation constants, KD, in the range from 25 to 41 nM, in a GM1 concentration-dependent manner. Biolayer interferometry experiments confirmed the high-affinity binding of monomeric Aß(1-40) (KD of 24 nM to 49 nM) as well as of Aß(1-42) (KD of 30 nM) to GM1 containing nanodiscs, and no binding to phospholipid containing nanodiscs. Interestingly, and in contrast to monomeric Aß, neither oligomeric Aß(1-40) nor oligomeric Aß(1-42) binds to GM1 nanodiscs. To the best of our knowledge, this is the first report of a loss of function for monomeric Aß upon aggregation.

The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif.

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na(+) channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain-ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83Å(2)) between R430 (WW3*) and L647' (αENaC). This corroborates the model-free analysis of the (15)N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S(2)=0.90±0.01). Carr-Purcell-Meiboom-Gill relaxation dispersion analysis identified two different conformational exchange processes on the µs-ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.

Phages have adapted the same protein fold to fulfill multiple functions in virion assembly.

Evolutionary relationships may exist among very diverse groups of proteins even though they perform different functions and display little sequence similarity. The tailed bacteriophages present a uniquely amenable system for identifying such groups because of their huge diversity yet conserved genome structures. In this work, we used structural, functional, and genomic context comparisons to conclude that the head-tail connector protein and tail tube protein of bacteriophage lambda diverged from a common ancestral protein. Further comparisons of tertiary and quaternary structures indicate that the baseplate hub and tail terminator proteins of bacteriophage may also be part of this same family. We propose that all of these proteins evolved from a single ancestral tail tube protein fold, and that gene duplication followed by differentiation led to the specialized roles of these proteins seen in bacteriophages today. Although this type of evolutionary mechanism has been proposed for other systems, our work provides an evolutionary mechanism for a group of proteins with different functions that bear no sequence similarity. Our data also indicate that the addition of a structural element at the N terminus of the lambda head-tail connector protein endows it with a distinctive protein interaction capability compared with many of its putative homologues.

Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding.

Many experimental and theoretical studies have suggested a significant role for nonnative interactions in protein folding pathways, but the energetic contributions of these interactions are not well understood. We have addressed the energetics and the position specificity of nonnative hydrophobic interactions by developing a continuum coarse-grained chain model with a native-centric potential augmented by sequence-dependent hydrophobic interactions. By modeling the effect of different hydrophobicity values at various positions in the Fyn SH3 domain, we predicted energetically significant nonnative interactions that led to acceleration or deceleration of the folding rate depending on whether they were more populated in the transition state or unfolded state. These nonnative contacts were centered on position 53 in the Fyn SH3 domain, which lies in an exposed position in a 3(10)-helix. The energetic importance of the predicted nonnative interactions was confirmed experimentally by folding kinetics studies combined with double mutant thermodynamic cycles. By attaining agreement of theoretical and experimental investigations, this study provides a compelling demonstration that specific nonnative interactions can significantly influence folding energetics. Moreover, we show that a coarse-grained model with a simple consideration of hydrophobicity is sufficient for the accurate prediction of kinetically important nonnative interactions.

Determination of isoleucine side-chain conformations in ground and excited states of proteins from chemical shifts.

A simple method is presented for quantifying Ile chi(2) rotamer distributions in proteins based on the measurement of Ile (13)C(delta1) chemical shifts. The methodology is well suited for applications involving very high molecular weight protein complexes, where other NMR parameters such as side-chain scalar coupling constants that report on dihedral angles cannot be measured or for studies of invisible, excited protein states, where chemical shifts are obtained from analysis of CPMG relaxation dispersion profiles. The utility of the approach is demonstrated by an application to the folding reaction of a mutant Fyn SH3 domain, where Ile side-chain structure and dynamics of an on-folding pathway intermediate state are studied.

Structural properties and dynamic behavior of nonfibrillar oligomers formed by PrP(106-126).

The formation of nonfibrillar oligomers has been proposed as a common element of the aggregation pathway of proteins and peptides associated with neurodegenerative diseases such as Alzheimer's and Creutzfeldt-Jakob disease. While fibrillar structures have long been considered indicators of diseases linked with the accumulation of amyloid plaques, it has more recently been proposed that amyloid oligomers are in fact the cytotoxic form. Here we describe the local structure and dynamics of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). Structural constraints from solid-state NMR reveal quaternary packing interactions within the hydrophobic core, similar to those previously reported for amyloid fibrils formed by this peptide, and consistent with structural studies of oligomers formed by the Alzheimer's beta-amyloid peptide. However, a hydration-dependent increase in disorder is observed for nonfibrillar oligomers of PrP(106-126). In solution NMR spectra we observe narrow (1)H and (13)C resonances corresponding to a monomer in exchange with the approximately 30 nm diameter nonfibrillar oligomers, giving additional information on the molecular structure of these species. Taken together, our data support a model in which the local structure of the oligomers contains the basic elements of amyloid fibrils, but with long-range disorder and local mobility that distinguishes these assemblies from the fibrillar form of PrP(106-126). These characteristics may provide a basis for the differing biological activities of amyloid fibrils and oligomers.

Determination of Leu side-chain conformations in excited protein states by NMR relaxation dispersion.

Fits of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion profiles allow extraction of the kinetics and thermodynamics of exchange reactions that interconvert highly populated, ground state and low populated, excited state conformers. Structural information is also available in the form of chemical shift differences between the interconverting protein states. Here we present a very simple method for extracting chi(2) rotamer distributions of Leu side chains in 'invisible' excited protein states based on measurement of their (13)C(delta1)/(13)C(delta2) chemical shifts using methyl CPMG dispersion experiments. The methodology is applied to study the protein folding reaction of the Fyn SH3 domain. A uniform chi(2) rotamer distribution is obtained for Leu residues of the unfolded state, with each Leu occupying the trans and gauche+ conformations in a 2:1 ratio. By contrast, leucines of an 'invisible' Fyn SH3 domain folding intermediate show a much more heterogeneous distribution of chi(2) rotamer populations. The experiment provides an important tool toward the quantitative characterization of both the structural and dynamics properties of states that cannot be studied by other biophysical tools.

Mutational investigation of protein folding transition states by Phi-value analysis and beyond: lessons from SH3 domain folding.

Understanding how proteins adopt their unique native structures requires a complete structural characterization of the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly populated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid substitutions (especially to Ala) via Phi-value analysis is the most common method to probe the structure of these transient, yet important states. A critical review of the key assumptions required for rigorous interpretation of Phi values reveals that a multiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively to Ala, provides the best means to characterize folding transition states. This approach has proven useful in revealing non-native interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the folding kinetics of multiple substitutions made at a single surface-exposed position using the Brønsted analysis the backbone conformation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milliseconds, the kinetic parameters for Phi-value analysis can be obtained from NMR relaxation dispersion experiments, under fully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native, denatured, and intermediate states that populate the pathway). This additional structural information, which is not readily obtained through stopped-flow based methods, can significantly facilitate the interpretation of Phi values because it often reports on the validity of the assumptions required for a rigorous interpretation of Phi values.

A simple method for measuring signs of (1)H (N) chemical shift differences between ground and excited protein states.

NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible, ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values of the chemical shift differences between interconverting states, /Delta(omega)/, opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the availability of the chemical shifts of the excited state that can only be obtained once the signs of Delta(omega) are known. Herein we describe a very simple method for determining the signs of (1)H(N) Delta(omega) values based on a comparison of peak positions in the directly detected dimensions of a pair of (1)H(N)-(15)N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other experiments, such as those required for measuring signs of (15)N Delta(omega) values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches.

Measurement of signs of chemical shift differences between ground and excited protein states: a comparison between H(S/M)QC and R1rho methods.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible 'excited' conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Deltavarpi|), that inform on the structural properties of the excited state(s). The sign of Deltavarpi is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of (1)H(N) and (13)C(alpha) Deltavarpi values using weak off-resonance R (1rho ) relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of (15)N and (1)H(alpha) shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Deltavarpi values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone.

Clustering of human prion protein and α-synuclein oligomers requires the prion protein N-terminus.

The interaction of prion protein (PrP) and α-synuclein (αSyn) oligomers causes synaptic impairment that might trigger Parkinson's disease and other synucleinopathies. Here, we report that αSyn oligomers (αSynO) cluster with human PrP (huPrP) into micron-sized condensates. Multivalency of αSyn within oligomers is required for condensation, since clustering with huPrP is not observed for monomeric αSyn. The stoichiometry of the heteroassemblies is well defined with an αSyn:huPrP molar ratio of about 1:1. The αSynO-huPrP interaction is of high affinity, signified by slow dissociation. The huPrP region responsible for condensation of αSynO, residues 95-111 in the intrinsically disordered N-terminus, corresponds to the region required for αSynO-mediated cognitive impairment. HuPrP, moreover, achieves co-clustering of αSynO and Alzheimer's disease-associated amyloid-ß oligomers, providing a case of a cross-interaction of two amyloidogenic proteins through an interlinking intrinsically disordered protein region. The results suggest that αSynO-mediated condensation of huPrP is involved in the pathogenesis of synucleinopathies.

TopModel: Template-Based Protein Structure Prediction at Low Sequence Identity Using Top-Down Consensus and Deep Neural Networks.

Knowledge of protein structures is essential to understand proteins' functions, evolution, dynamics, stabilities, and interactions and for data-driven protein- or drug design. Yet, experimental structure determination rates are far exceeded by that of next-generation sequencing, resulting in less than 1/1000th of proteins having an experimentally known 3D structure. Computational structure prediction seeks to alleviate this problem, and the Critical Assessment of Protein Structure Prediction (CASP) has shown the value of consensus and meta-methods that utilize complementary algorithms. However, traditionally, such methods employ majority voting during template selection and model averaging during refinement, which can drive the model away from the native fold if it is underrepresented in the ensemble. Here, we present TopModel, a fully automated meta-method for protein structure prediction. In contrast to traditional consensus and meta-methods, TopModel uses top-down consensus and deep neural networks to select templates and identify and correct wrongly modeled regions. TopModel combines a broad range of state-of-the-art methods for threading, alignment, and model quality estimation and provides a versatile workflow and toolbox for template-based structure prediction. TopModel shows a superior template selection, alignment accuracy, and model quality for template-based structure prediction on the CASP10-12 datasets compared to 12 state-of-the-art stand-alone primary predictors. TopModel was validated by prospective predictions of the nisin resistance protein (NSR) protein from Streptococcus agalactiae and LipoP from Clostridium difficile, showing far better agreement with experimental data than any of its constituent primary predictors. These results, in general, demonstrate the utility of TopModel for protein structure prediction and, in particular, show how combining computational structure prediction with sparse or low-resolution experimental data can improve the final model.

Relaxation dispersion NMR spectroscopy as a tool for detailed studies of protein folding.

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.

Measuring the signs of 1H(alpha) chemical shift differences between ground and excited protein states by off-resonance spin-lock R(1rho) NMR spectroscopy.

Analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical shift differences between NMR probes in the exchanging states, |Delta omega|, are also extracted. Herein, we present a simple experiment for obtaining the sign of (1)H(alpha) Delta omega values by measuring off-resonance (1)H(alpha) decay rates, R(1rho), using weak proton spin-lock fields. A pair of R(1rho) values is measured with a spin-lock field applied |Delta omega| downfield and upfield of the major-state peak. In many cases, these two relaxation rates differ substantially, with the larger one corresponding to the case where the spin-lock field coincides with the resonance frequency of the probe in the minor state. The utility of the methodology is demonstrated first on a system involving protein ligand exchange and subsequently on an SH3 domain exchanging between a folded state and its on-pathway folding intermediate. With this experiment, it thus becomes possible to determine (1)H(alpha) chemical shifts of the invisible excited state, which can be used as powerful restraints in defining the structural properties of these elusive conformers.