Advances in NMR Spectroscopy of Weakly Aligned Biomolecular Systems.

The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weaknesses. In parallel, an enormous number of pulse schemes have been introduced to measure the many different types of RDCs, ranging from the most widely measured backbone amide 15N-1H RDCs, to 1H-1H RDCs and couplings between low-γ nuclei. Applications of RDCs range from structure validation and refinement to the determination of relative domain orientations, the measurement of backbone and domain motions, and de novo structure determination. Nevertheless, it appears that the power of the RDC methodology remains underutilized. This review aims to highlight the practical aspects of sample preparation and RDC measurement while describing some of the most straightforward applications that take advantage of the exceptionally precise information contained in such data. Some emphasis will be placed on more recent developments that enable the accurate measurement of RDCs in larger systems, which is key to the ongoing shift in focus of biological NMR spectroscopy from structure determination toward gaining improved understanding of how molecular flexibility drives protein function.

Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy.

Although the α-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a protein's structure. Highly charged single α-helical (SAH) domains, consisting of a high percentage (>75%) of Arg, Lys, and Glu residues, are exceptions to this rule but have been difficult to characterize structurally. Our study focuses on the 68-residue medial tail domain of myosin-VI, which is found to contain a highly ordered α-helical structure extending from Glu-6 to Lys-63. High hydrogen exchange protection factors (15-150), small (ca. 4 Hz) 3 JHNHα couplings, and a near-perfect fit to an ideal model α-helix for its residual dipolar couplings (RDCs), measured in a filamentous phage medium, support the high regularity of this helix. Remarkably, the hydrogen exchange rates are far more homogeneous than the protection factors derived from them, suggesting that for these transiently broken helices the intrinsic exchange rates derived from the amino acid sequence are not appropriate reference values. 15N relaxation data indicate a very high degree of rotational diffusion anisotropy ( D∥/ D⊥ ≈ 7.6), consistent with the hydrodynamic behavior predicted for such a long, nearly straight α-helix. Alignment of the helix by a paramagnetic lanthanide ion attached to its N-terminal region shows a decrease in alignment as the distance from the tagging site increases. This decrease yields a precise measure for the persistence length of 224 ± 10 Å at 20 °C, supporting the idea that the role of the SAH helix is to act as an extension of the myosin-VI lever arm.

Monitoring Hydrogen Exchange During Protein Folding by Fast Pressure Jump NMR Spectroscopy.

A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a pressure-sensitized ubiquitin variant that contains two Val to Ala mutations. Increased protection against hydrogen exchange with solvent is monitored as a function of time during the folding process. Results for 53 backbone amides show narrow clustering with protection occurring with a time constant of ca. 85 ms, but slower protection is observed around a reverse turn near the C-terminus of the protein. Remarkably, the native NMR spectrum returns with this slower time constant of ca. 150 ms, indicating that the almost fully folded protein retains molten globule characteristics with severe NMR line broadening until the final hydrogen bonds are formed. Prior to crossing the transition state barrier, hydrogen exchange protection factors are close to unity, but with slightly elevated values in the ß1-ß2 hairpin, previously shown to be already lowly populated in the urea-denatured state.

Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data.

Implementation of a new algorithm, SMILE, is described for reconstruction of non-uniformly sampled two-, three- and four-dimensional NMR data, which takes advantage of the known phases of the NMR spectrum and the exponential decay of underlying time domain signals. The method is very robust with respect to the chosen sampling protocol and, in its default mode, also extends the truncated time domain signals by a modest amount of non-sampled zeros. SMILE can likewise be used to extend conventional uniformly sampled data, as an effective multidimensional alternative to linear prediction. The program is provided as a plug-in to the widely used NMRPipe software suite, and can be used with default parameters for mainstream application, or with user control over the iterative process to possibly further improve reconstruction quality and to lower the demand on computational resources. For large data sets, the method is robust and demonstrated for sparsities down to ca 1%, and final all-real spectral sizes as large as 300 Gb. Comparison between fully sampled, conventionally processed spectra and randomly selected NUS subsets of this data shows that the reconstruction quality approaches the theoretical limit in terms of peak position fidelity and intensity. SMILE essentially removes the noise-like appearance associated with the point-spread function of signals that are a default of five-fold above the noise level, but impacts the actual thermal noise in the NMR spectra only minimally. Therefore, the appearance and interpretation of SMILE-reconstructed spectra is very similar to that of fully sampled spectra generated by Fourier transformation.

ARTSY-J: Convenient and precise measurement of (3)JHNHα couplings in medium-size proteins from TROSY-HSQC spectra.

A new and convenient method, named ARTSY-J, is introduced that permits extraction of the (3)JHNHα couplings in proteins from the relative intensities in a pair of (15)N-(1)H TROSY-HSQC spectra. The pulse scheme includes (3)JHNHα dephasing of the narrower TROSY (1)H(N)-{(15)N} doublet component during a delay, integrated into the regular two-dimensional TROSY-HSQC pulse scheme, and compares the obtained intensity with a reference spectrum where (3)JHNHα dephasing is suppressed. The effect of passive (1)H(α) spin flips downscales the apparent (3)JHNHα coupling by a uniform factor that depends approximately linearly on both the duration of the (3)JHNHα dephasing delay and the (1)H-(1)H cross relaxation rate. Using such a correction factor, which accounts for the effects of both inhomogeneity of the radiofrequency field and (1)H(α) spin flips, agreement between prior and newly measured values for the small model protein GB3 is better than 0.3Hz. Measurement for the HIV-1 protease homodimer (22kDa) yields (3)JHNHα values that agree to better than 0.7Hz with predictions made on the basis of a previously parameterized Karplus equation. Although for Gly residues the two individual (3)JHNHα couplings cannot be extracted from a single set of ARTSY-J spectra, the measurement provides valuable ϕ angle information.

NMR studies of dynamic biomolecular conformational ensembles.

Multidimensional heteronuclear NMR approaches can provide nearly complete sequential signal assignments of isotopically enriched biomolecules. The availability of assignments together with measurements of spin relaxation rates, residual spin interactions, J-couplings and chemical shifts provides information at atomic resolution about internal dynamics on timescales ranging from ps to ms, both in solution and in the solid state. However, due to the complexity of biomolecules, it is not possible to extract a unique atomic-resolution description of biomolecular motions even from extensive NMR data when many conformations are sampled on multiple timescales. For this reason, powerful computational approaches are increasingly applied to large NMR data sets to elucidate conformational ensembles sampled by biomolecules. In the past decade, considerable attention has been directed at an important class of biomolecules that function by binding to a wide variety of target molecules. Questions of current interest are: "Does the free biomolecule sample a conformational ensemble that encompasses the conformations found when it binds to various targets; and if so, on what time scale is the ensemble sampled?" This article reviews recent efforts to answer these questions, with a focus on comparing ensembles obtained for the same biomolecules by different investigators. A detailed comparison of results obtained is provided for three biomolecules: ubiquitin, calmodulin and the HIV-1 trans-activation response RNA.

Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy.

We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. (15)N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR(2)) in (15)N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The (15)N-DEST and ΔR(2) data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-ß (Aß) at concentrations of ~100 µM.

Atomic-resolution dynamics on the surface of amyloid-ß protofibrils probed by solution NMR.

Exchange dynamics between molecules free in solution and bound to the surface of a large supramolecular structure, a polymer, a membrane or solid support are important in many phenomena in biology and materials science. Here we present a novel and generally applicable solution NMR technique, known as dark-state exchange saturation transfer (DEST), to probe such exchange phenomena with atomic resolution. This is illustrated by the exchange reaction between amyloid-ß (Aß) monomers and polydisperse, NMR-invisible ('dark') protofibrils, a process of significant interest because the accumulation of toxic, aggregated forms of Aß, from small oligomers to very large assemblies, has been implicated in the aetiology of Alzheimer's disease. The (15)N-DEST experiment imprints with single-residue-resolution dynamic information on the protofibril-bound species in the form of (15)N transverse relaxation rates ((15)N-R(2)) and exchange kinetics between monomers and protofibrils onto the easily observed two-dimensional (1)H-(15)N correlation spectrum of the monomer. The exchanging species on the protofibril surface comprise an ensemble of sparsely populated states where each residue is either tethered to (through other residues) or in direct contact with the surface. The first eight residues exist predominantly in a mobile tethered state, whereas the largely hydrophobic central region and part of the carboxy (C)-terminal hydrophobic region are in direct contact with the protofibril surface for a significant proportion of the time. The C-terminal residues of both Aß40 and Aß42 display lower affinity for the protofibril surface, indicating that they are likely to be surface exposed rather than buried as in structures of Aß fibrils, and might therefore comprise the critical nucleus for fibril formation. The values, however, are significantly larger for the C-terminal residues of Aß42 than Aß40, which might explain the former's higher propensity for rapid aggregation and fibril formation.

Dynamics of biomolecules from picoseconds to seconds at atomic resolution.

Although biomolecular dynamics has been investigated using NMR for at least 40 years, only in the past 20 years have internal motions been characterized at atomic resolution throughout proteins and nucleic acids. This development was made possible by multidimensional heteronuclear NMR approaches that provide near complete sequential signal assignments of uniformly labeled biomolecules. Recent methodological advances have enabled characterization of internal dynamics on timescales ranging from picoseconds to seconds, both in solution and in the solid state. The size, complexity and functional significance of biomolecules investigated by NMR continue to grow, as do the insights that have been obtained about function. In this article I review a number of recent advances that have made such studies possible, and provide a few examples of where NMR either by itself or in combination with other approaches has paved the way to a better understanding of the complex relationship between dynamics and biomolecular function. Finally, I discuss prospects for further advances in this field.

Measuring rapid hydrogen exchange in the homodimeric 36 kDa HIV-1 integrase catalytic core domain.

Measurements of rapid hydrogen exchange (HX) of water with protein amide sites contain valuable information on protein structure and function, but current NMR methods for measuring HX rates are limited in their applicability to large protein systems. An alternate method for measuring rapid HX is presented that is well-suited for larger proteins, and we apply the method to the deuterated, homodimeric 36 kDa HIV-1 integrase catalytic core domain (CCD). Using long mixing times for water-amide magnetization exchange at multiple pH values, HX rates spanning more than four orders of magnitude were measured, as well as NOE cross-relaxation rates to nearby exchangeable protons. HX protection factors for the CCD are found to be large (>10(4)) for residues along the dimer interface, but much smaller in many other regions. Notably, the catalytic helix (residues 152-167) exhibits low HX protection at both ends, indicative of fraying at both termini as opposed to just the N-terminal end, as originally thought. Residues in the LEDGF/p75 binding pocket also show marginal stability, with protection factors in the 10-100 range (∼1.4-2.7 kcal/mol). Additionally, elevated NOE cross-relaxation rates are identified and, as expected, correspond to proximity of the amide proton to a rapidly exchanging proton, typically from an OH side chain. Indirect NOE transfer between H(2) O and the amide proton of I141, a residue in the partially disordered active site of the enzyme, suggests its proximity to the side chain of S147, an interaction seen in the DNA-bound form for a homologous integrase.

Kinetics of amyloid beta monomer-to-oligomer exchange by NMR relaxation.

Recent studies have implicated non-fibrillar oligomers of the amyloid beta (Abeta) peptide as the primary toxic species in Alzheimer's disease. Detailed structural and kinetic characterization of these states, however, has been difficult. Here we use NMR relaxation measurements to address the kinetics of exchange between monomeric and large, polymorphic oligomeric species of Abeta(1-40). (15)N and (1)H(N) R(2) data at multiple magnetic fields were recorded for several peptide concentrations subsequent to the establishment of a stable pseudo-equilibrium between monomeric and NMR-invisible soluble oligomeric species. The increase in (15)N and (1)H(N) R(2) rates as a function of protein concentration is independent of nucleus and magnetic field and shows only a small degree of variation along the peptide chain. This phenomenon is due to a lifetime broadening effect arising from the unidirectional conversion of monomer to the NMR-invisible oligomeric species ("dark" state). At a total Abeta(1-40) concentration of 300 microM, the apparent first-order rate constant for this process is approximately 3 s(-1). Fitting the McConnell equations for two dipolar-coupled spins in two-site exchange to transfer-of-saturation profiles at two radiofrequency field strengths gives an estimate for k(off) of 73 s(-1) and transiently bound monomer (1)H(N) R(2) rates of up to 42,000 s(-1) in the tightly bound central hydrophobic region and approximately 300 s(-1) in the disordered regions, such as the first nine residues. The fraction of peptide within the "dark" oligomeric state undergoing exchange with free monomer is calculated to be approximately 3%.

Slight mistuning of a cryogenic probe significantly perturbs the water 1H precession frequency.

A shift of the water proton precession frequency is described that can introduce errors in chemical shifts derived using the water signal as the chemical shift reference. This shift, f(s), arises as a consequence of radiation damping when the water proton and detector circuit resonance frequencies differ. Herein it is shown that experimental values of f(s), measured as a function of detector circuit tuning offset for 500 and 900 MHz cryogenic probes, are in good agreement with theory. Of importance is the fact that even a small degree of mistuning, which does not significantly impact the performance of a pulse sequence, introduces chemical shift errors of +/-0.03 ppm, that negatively impact many types of experiments. A simple remedy that attenuates the frequency shift is presented.

Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis.

A novel iterative procedure is described that allows both the orientation and dynamics of internuclear bond vectors to be determined from direct interpretation of NMR dipolar couplings, measured under at least three orthogonal alignment conditions. If five orthogonal alignments are available, the approach also yields information on the degree of motional anisotropy and the direction in which the largest amplitude internal motion of each bond vector takes place. The method is demonstrated for the backbone (15)N-(1)H, (13)C(alpha)-(1)H(alpha), and (13)C(alpha)-13C' interactions in the previously well-studied protein domain GB3, dissolved in a liquid crystalline suspension of filamentous phage Pf1. Alignment variation is achieved by using conservative mutations of charged surface residues. Results indicate remarkably uniform backbone dynamics, with amplitudes that agree well with those of previous (15)N relaxation studies for most residues involved in elements of secondary structure, but larger amplitude dynamics than those found by (15)N relaxation for residues in loop and turn regions. In agreement with a previous analysis of dipolar couplings, the N-H bonds in the second beta-strand, which is involved in antibody recognition, show elevated dynamics with largest amplitudes orthogonal to the chain direction.

Mutational and structural studies aimed at characterizing the monomer of HIV-1 protease and its precursor.

An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of approximately 20 micoM. Under these conditions, NMR spectra show that the mature protease lacking its terminal beta-sheet residues 1-4 and 96-99 (PR(5-95)) exhibits a stable monomer fold spanning the region 10-90 that is similar to that of the single subunit of the wild-type dimer and the dimer bearing a D25N mutation (PR(D25N)). Urea-induced unfolding monitored both by changes in (1)H-(15)N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR(5-95) monomer displays a transition profile similar to that of the PR(D25N) dimer (50% unfolded (U(50)) = approximately 1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ((SFNF)PR(D25N)) decreases the stability of the fold (U(50) = approximately 1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR(T26A) (U(50) = 2.5 M) and (SFNF)PR(D25N) monomers and compared with PR(D25N/T26A) monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of (SFNF)PR(D25N) that may relate to the increase in the equilibrium dissociation constant (K(d)) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.

Accuracy of optimized chemical-exchange parameters derived by fitting CPMG R2 dispersion profiles when R2(0a) not = R2(0b).

The transverse relaxation rate, R2, measured as a function of the effective field (R2 dispersion) using a Carr-Purcell-Meiboom-Gill (CPMG) pulse train, is well suited to detect conformational exchange in proteins. The dispersion data are commonly fitted by a two-site (sites a and b) exchange model with four parameters: the relative population, pa, the difference in chemical shifts of the two sites, deltaomega, the correlation time for exchange, tau(ex), and the intrinsic relaxation rate (i.e., transverse relaxation rate in the absence of chemical exchange), R2(0). Although the intrinsic relaxation rates of the two sites, R2(0a) and R2(0b), can differ, they are normally assumed to be the same (i.e., R2(0a) = R2(0b) = R2(0)) when fitting dispersion data. The purpose of this investigation is to determine the magnitudes of the errors in the optimized exchange parameters that are introduced by the assumption that R2(0a) = R2(0b). In order to accomplish this goal, we first generated synthetic constant-time CPMG R2 dispersion data assuming two-site exchange with R2(0a) not equal R2(0b), and then fitted the synthetic data assuming two-site exchange with R2(0) = R2(0a) = R2(0b). Although all the synthetic data generated assuming R2(0a) not equal R2(0b) were well fitted (assuming R2(0a) = R2(0b)), the optimized values of pa and tau(ex) differed from their true values, whereas the optimized values of deltaomega values did not. A theoretical analysis using the Carver-Richards equation explains these results, and yields simple, general equations for estimating the magnitudes of the errors in the optimized parameters, as a function of (R2(0a) - R2(0b)).

Error estimation and global fitting in transverse-relaxation dispersion experiments to determine chemical-exchange parameters.

Off-resonance effects can introduce significant systematic errors in R2 measurements in constant-time Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion experiments. For an off-resonance chemical shift of 500 Hz, 15N relaxation dispersion profiles obtained from experiment and computer simulation indicated a systematic error of ca. 3%. This error is three- to five-fold larger than the random error in R2 caused by noise. Good estimates of total R2 uncertainty are critical in order to obtain accurate estimates in optimized chemical exchange parameters and their uncertainties derived from chi2 minimization of a target function. Here, we present a simple empirical approach that provides a good estimate of the total error (systematic + random) in 15N R2 values measured for the HIV protease. The advantage of this empirical error estimate is that it is applicable even when some of the factors that contribute to the off-resonance error are not known. These errors are incorporated into a chi2 minimization protocol, in which the Carver-Richards equation is used fit the observed R2 dispersion profiles, that yields optimized chemical exchange parameters and their confidence limits. Optimized parameters are also derived, using the same protein sample and data-fitting protocol, from 1H R2 measurements in which systematic errors are negligible. Although 1H and 15N relaxation profiles of individual residues were well fit, the optimized exchange parameters had large uncertainties (confidence limits). In contrast, when a single pair of exchange parameters (the exchange lifetime, tau(ex), and the fractional population, p(a)), were constrained to globally fit all R2 profiles for residues in the dimer interface of the protein, confidence limits were less than 8% for all optimized exchange parameters. In addition, F-tests showed that quality of the fits obtained using tau(ex), p(a) as global parameters were not improved when these parameters were free to fit the R2 profiles of individual residues. Finally, nearly the same optimized global tau(ex), p(a) values were obtained, when the 1H and 15N data sets for residues in the dimer interface, were fit independently; the difference in optimized global parameters, ca. 10%, was of marginal significance according to the F-test.

Carbonyl carbon transverse relaxation dispersion measurements and ms-micros timescale motion in a protein hydrogen bond network.

A constant-time, Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation, R(2), dispersion experiment for carbonyl carbons was designed and executed to detect micros-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) C(alpha)-C' J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly (13)C enriched proteins, unless care is taken to minimize the perturbation of the C(alpha) magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular (with an excitation null in the C(alpha) region of the spectrum) pulses were employed in order to minimize C' signal modulation by C(alpha)-C' J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [(13)C,(15)N, (2)H] ((2)H at non-labile hydrogen sites) labeled, and (2) uniformly (15)N/selectively-(13)C' labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly (13)C/(15)N/(2)H labeled sample was well suited to measure (15)N and (1)H R(2) dispersion as well as (13)C' dispersion, conformational exchange in the inter subunit beta-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.