Biomedical subjects

Nikolai R Skrynnikov

Publications and source records attributed to Nikolai R Skrynnikov.

14 recordsLinked to original sources

PubMed2006

Observation of microsecond time-scale protein dynamics in the presence of Ln3+ ions: application to the N-terminal domain of cardiac troponin C.

The microsecond time-scale motions in the N-terminal domain of cardiac troponin C (NcTnC) loaded with lanthanide ions have been investigated by means of a (1)H(N) off-resonance spin-lock experiment. The observed relaxation dispersion effects strongly increase along the series of NcTnC samples containing La(3+), Ce(3+), and Pr(3+) ions. This rise in dispersion effects is due to modulation of long-range pseudocontact shifts by micros time-scale dynamics. Specifically, the motion in the coordination sphere of the lanthanide ion (i.e. in the NcTnC EF-hand motif) causes modulation of the paramagnetic susceptibility tensor which, in turn, causes modulation of pseudocontact shifts. It is also probable that opening/closing dynamics, previously identified in Ca(2+)-NcTnC, contributes to some of the observed dispersions. On the other hand, it is unlikely that monomer-dimer exchange in the solution of NcTnC is directly responsible for the dispersion effects. Finally, on-off exchange of the lanthanide ion does not seem to play any significant role. The amplification of dispersion effects by Ln(3+) ions is a potentially useful tool for studies of micros-ms motions in proteins. This approach makes it possible to observe the dispersions even when the local environment of the reporting spin does not change. This happens, for example, when the motion involves a 'rigid' structural unit such as individual alpha-helix. Even more significantly, the dispersions based on pseudocontact shifts offer better chances for structural characterization of the dynamic species. This method can be generalized for a large class of applications via the use of specially designed lanthanide-binding tags.

Binding Sites↗

PubMed2006

Protein side-chain dynamics observed by solution- and solid-state NMR: comparative analysis of methyl 2H relaxation data.

Rapid advances in solid-state MAS NMR made it possible to probe protein dynamics on a per-residue basis, similar to solution experiments. In this work we compare methyl 2H relaxation rates measured in the solid and liquid samples of alpha-spectrin SH3 domain. The solution data are treated using a model-free approach to separate the contributions from the overall molecular tumbling and fast internal motion. The latter part forms the basis for comparison with the solid-state data. Although the accuracy of solid-state measurements is limited by deuterium spin diffusion, the results suggest a significant similarity between methyl dynamics in the two samples. This is a potentially important observation, preparing the ground for combined analysis of the dynamics data by solid- and solution-state NMR.

Animals↗

PubMed2006

Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex.

Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3* domain complexed to the second PY motif (227'TGLPSYDEALH237') of Comm. Unexpectedly, there are interactions between WW3* and ligand residues both N- and C-terminal to the PY motif. Residues Y232'-L236' form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3* responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.

Amino Acid Motifs↗

PubMed2005

A new spin probe of protein dynamics: nitrogen relaxation in 15N-2H amide groups.

(15)N spin relaxation data have provided a wealth of information on protein dynamics in solution. Standard R(1), R(1)(rho), and NOE experiments aimed at (15)N[(1)H] amide moieties are complemented in this work by HA(CACO)N-type experiments allowing the measurement of nitrogen R(1) and R(1)(rho) rates at deuterated (15)N[(2)D] sites. Difference rates obtained using this approach, R(1)((15)N[(1)H]) - R(1)((15)N[(2)D]) and R(2)((15)N[(1)H]) - R(2)((15)N[(2)D]), depend exclusively on dipolar interactions and are insensitive to (15)N CSA and R(ex) relaxation mechanisms. The methodology has been tested on a sample of peptostreptococcal protein L (63 residues) prepared in 50% H(2)O-50% D(2)O solvent. The results from the new and conventional experiments are found to be consistent, with respect to both local backbone dynamics and overall protein tumbling. Combining several data sets permits evaluation of the spectral density J(omega(D) + omega(N)) for each amide site. This spectral density samples a uniquely low frequency (26 MHz at a 500 MHz field) and, therefore, is expected to be highly useful for characterizing nanosecond time scale local motions. The spectral density mapping demonstrates that, in the case of protein L, J(omega(D) + omega(N)) values are compatible with the Lipari-Szabo interpretation of backbone dynamics based on the conventional (15)N relaxation data.

Amides↗

PubMed2005

A new amide proton R1rho experiment permits accurate characterization of microsecond time-scale conformational exchange.

A new off-resonance spin-lock experiment to record relaxation dispersion profiles of amide protons is presented. The sensitivity-enhanced HSQC-type sequence is designed to minimize the interference from cross-relaxation effects and ensure that the dispersion profiles in the absence of micros-ms time-scale dynamics are flat. Toward this end (i) the proton background is eliminated by sample deuteration (Ishima et al., 1998), (ii) 1H spin lock is applied to two-spin modes 2(H(x)Sin theta + H(z)Cos theta) N(z), and (iii) the tilt angle theta approximately 35 degrees is maintained throughout the series of measurements (Desvaux et al. Mol. Phys., 86 (1995) 1059). The relaxation dispersion profiles recorded in this manner sample a wide range of effective rf field strengths (up to and in excess of 20 kHz) which makes them particularly suitable for studies of motions on the time scale < or = 100 micros. The new experiment has been tested on the Ca2+-loaded regulatory domain of cardiac troponin C. Many residues show pronounced dispersions with remarkably similar correlation times of approximately 30 micros. Furthermore, these residues are localized in the regions that have been previously implicated in conformational changes (Spyracopoulos et al. Biochemistry, 36 (1997) 12138).

Amides↗

PubMed2005

Estimating the accuracy of protein structures using residual dipolar couplings.

It has been commonly recognized that residual dipolar coupling data provide a measure of quality for protein structures. To quantify this observation, a database of 100 single-domain proteins has been compiled where each protein was represented by two independently solved structures. Backbone 1H-15N dipolar couplings were simulated for the target structures and then fitted to the model structures. The fits were characterized by an R-factor which was corrected for the effects of non-uniform distribution of dipolar vectors on a unit sphere. The analyses show that favorable R values virtually guarantee high accuracy of the model structure (where accuracy is defined as the backbone coordinate rms deviation). On the other hand, unfavorable R values do not necessarily suggest low accuracy. Based on the simulated data, a simple empirical formula is proposed to estimate the accuracy of protein structures. The method is illustrated with a number of examples, including PDZ2 domain of human phosphatase hPTP1E.

Computer Simulation↗

PubMed2005

Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins.

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using 13CH3, 13CH2D, and 13CHD2 methyl isotopomers, is presented. For each labeling scheme, 1H-13C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with 13CH3, 13CH2D, and 13CHD2 labeling in Ile delta1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence's performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for 13CH3-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (13CH3 HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the 13CH3 HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both 13C and 1H spins. The HSQC and HMQC spectra of 13CH2D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, 13CH2D TROSY, that exploits cross-correlation effects to record 1H-13C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of 13CHD2-labeled methyl groups can be recorded with HSQC or HMQC; a new 13CHD2 HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the 13CH3 labeling strategy is the most beneficial from the perspective of sensitivity (gains > or =2.4 relative to either 13CH2D or 13CHD2 labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.

Carbon Isotopes↗

PubMed2004

Microsecond time-scale dynamics from relaxation in the rotating frame: experiments using spin lock with alternating phase.

A spin lock comprised of radiofrequency pulses with alternating phase, (x) (-x)(x) (-x) , is proposed as a new technique to probe microsecond time-scale dynamics. A series of R1rho measurements using different pulse duration tp allows one to determine exchange rate, kex, the product p(a)p(b)(Delta omega(ab))2 involving populations of the exchanging species, p(a) and p(b), together with chemical shift difference, (Delta omega(ab)), and the strength of the spin-lock field, B1. The interpretation is based on simple analytical expression for R1rho derived on the basis of Redfield theory. The application of the method is demonstrated for partially deuterated molecule of cyclohexane undergoing chair-to-chair interconversion at -9 degrees C.

Algorithms↗

PubMed2002

Detecting protein kinase recognition modes of calmodulin by residual dipolar couplings in solution NMR.

Calmodulin-regulated serine/threonine kinases (CaM kinases) play crucial roles in Ca2+-dependent signaling transduction pathways in eukaryotes. Despite having a similar overall molecular architecture of catalytic and regulatory domains, CaM kinases employ different binding modes for Ca2+/CaM recruitment which is required for their activation. Here we present a residual dipolar coupling (RDC)-based NMR approach to characterizing the molecular recognition of CaM with five different CaM kinases. Our analyses indicate that CaM kinase I and likely IV use the same CaM binding mode as myosin light chain kinase (1-14 motif), distinct from those of CaM kinase II (1-10 motif) and CaM kinase kinase (1-16- motif). This NMR approach provides an efficient experimental guide for homology modeling and structural characterization of CaM-target complexes.

Amino Acid Motifs↗

PubMed2002

Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation measurements employing trains of 180 degrees pulses with variable pulse spacing provide valuable information about systems undergoing millisecond-time-scale chemical exchange. Fits of the CPMG relaxation dispersion profiles yield rates of interconversion, relative populations, and absolute values of chemical shift differences between the exchanging states, |Deltaomega|. It is shown that the sign of Deltaomega that is lacking from CPMG dispersion experiments can be obtained from a comparison of chemical shifts in the indirect dimensions in either a pair of HSQC (heteronuclear single quantum coherence) spectra recorded at different magnetic fields or HSQC and HMQC (heteronuclear multiple quantum coherence) spectra obtained at a single field. The methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s(-1) at 25 degrees C.

Alanine↗

PubMed2002

An NMR experiment for the accurate measurement of heteronuclear spin-lock relaxation rates.

Rotating-frame relaxation rates, R(1)(rho), are often measured in NMR studies of protein dynamics. We show here that large systematic errors can be introduced into measured values of heteronuclear R(1)(rho) rates using schemes which are usually employed to suppress cross-correlation between dipole-dipole and CSA relaxation mechanisms. For example, in a scalar-coupled two-spin X-H spin system the use of (1)H WALTZ16 decoupling or (1)H pulses applied at regularly spaced intervals leads to a significant overestimation of heteronuclear R(1)(rho) values. The problem is studied experimentally and theoretically for (15)N-(1)H and (13)C-(1)H spin pairs, and simple schemes are described which eliminate the artifacts. The approaches suggested are essential replacements of existing methodology if accurate dynamics parameters are to be extracted from spin-lock relaxation data sets.

Bacterial Proteins↗

PubMed2002

Deuterium spin probes of side-chain dynamics in proteins. 1. Measurement of five relaxation rates per deuteron in (13)C-labeled and fractionally (2)H-enriched proteins in solution.

New pulse sequences are presented for the measurement of the relaxation of deuterium double quantum, quadrupolar order, and transverse antiphase magnetization in (13)CH(2)D methyl groups of (15)N-, (13)C-labeled, fractionally deuterated proteins. Together with previously developed experiments for measuring deuterium longitudinal and transverse decay rates [Muhandiram, D. R.; Yamazaki, T.; Sykes, B. D.; Kay, L. E. J. Am. Chem. Soc. 1995, 117, 11536], these schemes allow measurement of the five unique decay constants of a single deuteron, providing an unprecedented opportunity to investigate side-chain dynamics in proteins. All five deuterium relaxation rates have been measured for deuterons in the methyl groups of the B1 immunoglobulin binding domain of peptostreptococcal protein L and the N-terminal SH3 domain from the protein drk. Since values of the spectral density function at only three different frequencies contribute to the five relaxation rates, the self-consistency of the relaxation data is readily established. Very good agreement is obtained between calculated parameters describing the amplitudes and time scales of motion when different subsets of the relaxation data are employed.

Bacterial Proteins↗

PubMed2002

Deuterium spin probes of side-chain dynamics in proteins. 2. Spectral density mapping and identification of nanosecond time-scale side-chain motions.

In the previous paper in this issue we have demonstrated that it is possible to measure the five different relaxation rates of a deuteron in (13)CH(2)D methyl groups of (13)C-labeled, fractionally deuterated proteins. The extensive set of data acquired in these experiments provides an opportunity to investigate side-chain dynamics in proteins at a level of detail that heretofore was not possible. The data, acquired on the B1 domain of peptostreptococcal protein L, include 16 (9) relaxation measurements at 4 (2) different magnetic field strengths, 25 degrees C (5 degrees C). These data are shown to be self-consistent and are analyzed using a spectral density mapping procedure which allows extraction of values of the spectral density function at a number of frequencies with no assumptions about the underlying dynamics. Dynamics data from 31 of 35 methyls in the protein for which data could be obtained were well-fitted using the two-parameter Lipari-Szabo model (Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546). The data from the remaining 4 methyls can be fitted using a three-parameter version of the Lipari-Szabo model that takes into account, in a simple manner, additional nanosecond time-scale local dynamics. This interpretation is supported by analysis of a molecular dynamics trajectory where spectral density profiles calculated for side-chain methyl sites reflect the influence of slower (nanosecond) time-scale motions involving jumps between rotameric wells. A discussion of the minimum number of relaxation measurements that are necessary to extract the full complement of dynamics information is presented along with an interpretation of the extracted dynamics parameters.

Bacterial Proteins↗

PubMed2002

Calculations of the contribution of ring currents to the chemical shielding anisotropy.

Ring currents can exert a large effect upon the chemical shielding of NMR resonances. The analytical expression developed by Waugh and Fessenden (Waugh, J. S.; Fessenden, R. W. J. Am. Chem. Soc. 1957, 79, 846) and Johnson and Bovey (Johnson, C. E.; Bovey, F. A. J. Chem. Phys. 1957, 29, 1012) only quantifies the contribution of ring currents to the isotropic component of the shielding tensor. In the work described here an additional analytical expression is developed so that the contribution of ring currents to the full shielding tensor can be calculated, allowing an estimate of the influence of ring currents upon the chemical shielding anisotropy (CSA, Deltasigma). To test that this pair of analytical expressions can provide a reasonable estimate of the contribution of ring currents to the full shielding tensor a series of density functional calculations (DFT) were carried out. A shielding tensor in a model compound was calculated in two distinct ways. For the first series, DFT shielding calculations of the model compound were carried out in the presence of a benzene ring. For the second series a ring current contribution to the shielding tensor was calculated via the new expressions, and this was added to the result of a DFT shielding calculation which used in place of benzene the nonaromatic analogue 1,3 cyclohexadiene. The two series of results proved to be in excellent agreement. The pair of analytical expressions are used to calculate ring current contributions to the CSA (Deltasigma) of 1H(N) backbone amide resonances in a structure of the second type 2 module from the protein fibronectin. Significant CSA variations are predicted in particular for the 1H(N) of G42 which is most likely involved in a N-H...tpi aromatic hydrogen bond.

Journal Article↗