Biomedical subjects

Vladislav Yu Orekhov

Publications and source records attributed to Vladislav Yu Orekhov.

18 recordsLinked to original sources

PubMed2006

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form.

There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor zeta subunit. This domain exhibits specific dimerization that is distinct from non-specific aggregation behavior seen in many systems. In this work, we use diffusion and chemical shift mapping NMR data to show that the protein does not undergo a transition between disordered and ordered states upon dimerization. This finding opposes the generally accepted view on the behavior of intrinsically disordered proteins, provides evidence for the existence of specific dimerization interactions for intrinsically disordered protein species and opens a new line of research in this new and quickly developing field.

Dimerization↗

PubMed2006

Targeted acquisition for real-time NMR spectroscopy.

A target-oriented approach for the acquisition of information in biomolecular NMR spectroscopy is being developed. This approach combines concurrent data accumulation, processing, and monitoring of spectral quality. Real-time estimation of parameters allows acquisition to be stopped when results are complete and have a specified precision. The technique is based on multidimensional decomposition, which can process incomplete data. An incremental nonuniform sampling scheme ensures the optimization of resolution sensitivity. To validate this method, 3D HNCO spectra of three biomolecular systems (8 kDa ubiquitin, 22 kDa barstar-barnase complex, and 82 kDa malate synthase G) are processed incrementally at small acquisition time steps. The range of molecular sizes illustrates applicability in both sample- and sensitivity-limited regimes. In each case, the target was to acquire all backbone resonances in the spectra. For the three systems, the targets are achieved after 4.5 min, 1.6 h, and 22 h of acquisition time, respectively. A number of other targets that can be similarly monitored as a function of time are discussed.

Bacterial Proteins↗

PubMed2006

Removal of a time barrier for high-resolution multidimensional NMR spectroscopy.

We introduce the recursive multidimensional decomposition (R-MDD) method to speed recording of high-resolution NMR spectra. The measurement time is logarithmically dependent on the sizes of indirect spectral dimensions. R-MDD has the sensitivity and resolution advantages of optimized nonuniform acquisition schemes and is applicable to all types of biomolecular spectra. We demonstrated it for triple resonance experiments on three globular proteins (ubiquitin, azurin and the barstar-barnase complex) of 8-22 kDa.

Algorithms↗

PubMed2005

Multiple-site exchange in proteins studied with a suite of six NMR relaxation dispersion experiments: an application to the folding of a Fyn SH3 domain mutant.

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.

Deuterium Exchange Measurement↗

PubMed2005

High-resolution four-dimensional 1H-13C NOE spectroscopy using methyl-TROSY, sparse data acquisition, and multidimensional decomposition.

An approach for recording four-dimensional (4D) methyl (1)H-(13)C-(13)C-(1)H NOESY spectra with high resolution and sensitivity is presented and applied to Malate Synthase G (723 residues, 82 kDa). Sensitivity and resolution have been optimized using a highly deuterated, methyl-protonated sample in concert with methyl-TROSY, sparse data sampling in the three indirect dimensions, and 4D spectral reconstruction using multidimensional decomposition (MDD). A sparse data acquisition protocol is introduced that ensures that sufficiently long indirect acquisition times can be employed to exploit the decreased relaxation rates associated with methyl-TROSY, without increasing the duration of the 4D experiment beyond acceptable measurement times. In this manner, only a fraction ( approximately 30%) of the experimental data that would normally be needed to achieve a spectrum of high resolution is acquired. The reconstructed 4D spectrum is of similar resolution and sensitivity to three-dimensional (3D) (13)C-edited NOE spectra, is straightforward to analyze, and resolves ambiguities that emerge when 3D data sets only are considered.

Carbon Isotopes↗

PubMed2005

Off-resonance R(1rho) NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain.

An (15)N NMR R(1rho) relaxation experiment is presented for the measurement of millisecond time scale exchange processes in proteins. On- and off-resonance R(1rho) relaxation profiles are recorded one residue at a time using a series of one-dimensional experiments in concert with selective Hartmann-Hahn polarization transfers. The experiment can be performed using low spin-lock field strengths (values as low as 25 Hz have been tested), with excellent alignment of magnetization along the effective field achieved. Additionally, suppression of the effects of cross-correlated relaxation between dipolar and chemical shift anisotropy interactions and (1)H-(15)N scalar coupled evolution is straightforward to implement, independent of the strength of the (15)N spin-locking field. The methodology is applied to study the folding of a G48M mutant of the Fyn SH3 domain that has been characterized previously by CPMG dispersion experiments. It is demonstrated through experiment that off-resonance R(1rho) data measured at a single magnetic field and one or more spin-lock field strengths, with amplitudes on the order of the rate of exchange, allow a complete characterization of a two-site exchange process. This is possible even in the case of slow exchange on the NMR time scale, where complementary approaches involving CPMG-based experiments fail. Advantages of this methodology in relation to other approaches are described.

Nitrogen Isotopes↗

PubMed2005

Solution NMR-derived global fold of a monomeric 82-kDa enzyme.

The size of proteins that can be studied by solution NMR spectroscopy has increased significantly because of recent developments in methodology. Important experiments include those that make use of approaches that increase the lifetimes of NMR signals or that define the orientation of internuclear bond vectors with respect to a common molecular frame. The advances in NMR techniques are strongly coupled to isotope labeling methods that increase sensitivity and reduce the complexity of NMR spectra. We show that these developments can be exploited in structural studies of high-molecular-weight, single-polypeptide proteins, and we present the solution global fold of the monomeric 723-residue (82-kDa) enzyme malate synthase G from Escherichia coli, which has been extensively characterized by NMR in the past several years.

Enzymes↗

PubMed2005

Accuracy and robustness of three-way decomposition applied to NMR data.

Three-way decomposition is a very versatile analysis tool with applications in a variety of protein NMR fields. It has been used to extract structural data from 3D NOESYs, to determine relaxation rates in large proteins, to identify ligand binding in screening for lead compounds, and to complement non-uniformly recorded (sparse) spectra. All applications so far concerned experimental data sets; it thus remains to address questions of accuracy and robustness of the method using simulated data where the correct answer is known. Systematic tests are presented for relaxation and NOESY data sets. Mixtures of real and synthetic data are used to allow control of various parameters and comparisons with correct reference data, while working with input that is as realistic as possible. The influence of the following parameters is evaluated: signal-to-noise, overlap of signals and the use of a regularization procedure within the algorithm. The main criteria used for the evaluation are accuracy and precision. It is shown that deterioration of accuracy is indicated by internal checks such as decrease of precision. Both with relaxation data and when interpreting NOESY spectra, three-way decomposition exhibits a robust behavior in situations with severe signal overlap and/or poor signal-to-noise, e.g., by avoiding false positives in the NOE shapes of NOESY decompositions. As a complement to this study, three-way decomposition is compared to other methods that achieve the same type of results.

Algorithms↗

PubMed2004

Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures.

Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein. Following a careful model-free analysis, several protein regions with different dynamic regimes are identified. Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117. These findings are consistent with a gated electron transfer process according to the "dynamic docking" model. Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature. Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion. The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.

Azurin↗

PubMed2004

Double- and zero-quantum NMR relaxation dispersion experiments sampling millisecond time scale dynamics in proteins.

TROSY-based NMR relaxation dispersion experiments that measure the decay of double- and zero-quantum (1)H-(15)N coherences as a function of applied (1)H and (15)N radio frequency (rf) fields are presented for studying millisecond dynamic processes in proteins. These experiments are complementary to existing approaches that measure dispersions of single-quantum (15)N and (1)H magnetization. When combined, data from all four coherences provide a more quantitative picture of dynamics, making it possible to distinguish, for example, between two-site and more complex exchange processes. In addition, a TROSY-based pulse scheme is described for measuring the relaxation of amide (1)H single-quantum magnetization, obtained by a simple modification of the multiple-quantum experiments. The new methodology is applied to a point mutant of the Fyn SH3 domain that exchanges between folded and unfolded states at 25 degrees C.

Nuclear Magnetic Resonance, Biomolecular↗

PubMed2004

Accurate relaxation parameters for large proteins.

The practical applicability, performance, and robustness of three-way decomposition (TWD) for the extraction of relaxation parameters are demonstrated for a large protein with 370 residues, the maltose binding protein. An ordinary set of seven relaxation-modulated (15)N HSQC spectra, recorded at another site, is systematically analyzed. For all 341 assigned backbone amide groups, including 21 pairs and one group of three overlapped peaks, T1 decay values were determined. On isolated peaks, TWD extracts T1 values with systematically lower error bounds compared to conventional tools, although for these simple cases the improvements remain limited. However, in the presence of spectral artifacts, the decrease in errors can become significant, demonstrating the higher robustness of TWD. For about half of the peaks in overlapped regions, the decomposition allowed separation of the signals, yielding significantly different T1 values between overlapping signals. For the rest, similarity of the decay times for the two or three overlapping signals could be confirmed within usually low error bounds. The use of TWD thus leads to a significant increase in the number of accessible relaxation probes in large proteins. With a newly implemented graphical user interface, the application of TWD requires merely a peak list, and thus no additional effort compared to conventional approaches is needed.

Algorithms↗

PubMed2003

Off-resonance R1rho relaxation outside of the fast exchange limit: an experimental study of a cavity mutant of T4 lysozyme.

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.

Magnetic Resonance Spectroscopy↗

PubMed2003

Optimizing resolution in multidimensional NMR by three-way decomposition.

Resolution depends on the number of points sampled in a FID; in indirectly detected dimensions it is an important determinant of the total experiment time. Based on the high redundancy present in NMR data, we propose the following timesaving scheme for three-dimensional spectra. An extensive grid of discrete t1- and t2-values is used, which increases resolution while preserving the spectral width. Total experiment time is reduced by avoiding the recording of t3-FIDs for selected pairs of t1 and t2; typically the recording is omitted for about 75% of the (t1,t2) combinations. These data sets are referred to as sparse, and post-experimental processing making optimal use of spectral redundancy provides the missing, non-recorded data. We have previously shown that three-way decomposition (TWD) within the MUNIN approach provides a practical way to process dense NMR data sets. Here, a novel TWD algorithm [Ibraghimov, (2002) Numer. Linear Algebra Appl. 9, 551-565] is used to complement a sparsely recorded time-domain data set by providing the missing FIDs for all (t1,t2) combinations omitted in the experiment. A necessary condition is that for each t1-value at least a few FIDs are recorded, and similar for each t2-value. The method is demonstrated on non-uniformly sampled (15)N-NOESY-HSQC data sets recorded for the 14 kD protein azurin. The spectra obtained by TWD, reconstruction and ordinary transform to frequency-domain are, in spite of the large number of signals and the high dynamic range typical for NOESYs, highly similar to a corresponding reference spectrum, for which all (t1,t2) combinations were recorded.

Magnetic Resonance Spectroscopy↗

PubMed2003

NMR detection of multiple transitions to low-populated states in azurin.

Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone (15)N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8 degrees C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of (15)N nuclei involved in the faster process exhibits at 44.8 degrees C a forward rate constant of 11.7+/-2.4 s(-1) and a population of the exited state of 0.39+/-0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8 degrees C are 4.1+/-0.1 s(-1) and 0.45+/-0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 A, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy-dominated transition around the copper, but an entropy-controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy-cost rearrangement of the copper-binding site that may be necessary for efficient electron transfer.

Azurin↗

PubMed2002

Automated analysis of large sets of heteronuclear correlation spectra in NMR-based drug discovery.

Drug discovery procedures based on NMR typically require the analysis of thousands of NMR spectra. For example, in "SAR by NMR", two-dimensional NMR spectra are recorded for a target protein mixed with ligand candidates from a comprehensive library of small molecules and are compared to the corresponding spectrum for the protein alone. We present an automated procedure for the comparative analysis of large sets of heteronuclear single quantum coherence spectra, which is based on three-way decomposition and implemented as the software package MUNIN. In a single step, spectra with differences in the peak positions (indicating ligand binding) and the affected peaks are identified. By omission of peak picking, ad hoc scoring of the quality of doubtful peaks is avoided. The procedure has been tested on the bacterial ribonuclease barnase, with a protein concentration of only 50 microM, using several small molecules including the substrate analogue 3'-GMP. Sets of 51 spectra were processed simultaneously, and it is concluded that spectra with binding ligands can be unambiguously identified from much larger sets of spectra.

Bacterial Proteins↗

PubMed2002

Dynamics-modulated biological activity of transforming growth factor beta3.

Transforming growth factor beta3 (TGF-beta3) is an important mediator of growth, maintenance, and repair processes in human cells. Internal dynamic properties have been derived from (15)N NMR relaxation data and mapped onto the spatial structure of TGF-beta3. The pattern of internal dynamics in the structure identifies potential "hot spots" of binding free energy and reveals the importance of conformational entropy in the interaction of TGF-beta3 with the receptors. The observed internal dynamics set TGF-beta3 apart from other TGF-beta isoforms, with which it shares the same fold. These findings may explain functional differences among the various TGF-beta isoforms and thus prove essential in the search for related therapeutic agents.

Amino Acid Sequence↗

PubMed2002

Essential domain motions in barnase revealed by MD simulations.

The wealth of data accumulated on the bacterial ribonuclease barnase is complemented by molecular dynamics trajectories starting from four different experimental structures and covering a total of >10 ns. Using principal component analysis, the simulations are interpreted in view of dynamic domains and hinges promoting relative motions of these domains. Two domains with residues 7-22 and 52-108 for the first domain and residues 25-51 for the second domain were consistently observed. Hinge regions consist primarily of Tyr24, Ser50, Ile51, and Gly52. Earlier mutation studies have demonstrated that the residues of the hinge regions play essential roles for the stability and activity of barnase. The domain motions are correlated to inter-domain interactions involving functionally important active site residues, such as Lys27 and Glu73. A model is presented that combines the observation of dynamic domains and their motions with the extensive mutation data from the literature. Enthalpic energy contributions originating from specific inter-domain interactions as well as entropic energy contributions due to the domain motions are discussed in the frame of this model and compared with destabilization energies measured for corresponding mutants.

Bacterial Proteins↗

PubMed2002

Three-way decomposition of a complete 3D 15N-NOESY-HSQC.

Three-way decomposition is applied for the structural analysis of a complete three-dimensional (15)N-NOESY-HSQC of the 128 residues long protein azurin. The procedure presented includes decomposition using the software MUNIN, providing an initial characterization of the complete spectrum by 355 components. This is followed by post-processing yielding a final list of 149 components, 123 of which characterize 1859 NOE peaks from backbone N-H groups. Components from three-way decomposition are defined as direct products of one-dimensional shapes along the three dimensions. Thus, a complete set of distance constraints from this spectrum is obtained by one-dimensional peak picking of the shapes along the NOE dimension. Correctness and completeness of this set of NOEs are tested for all backbone amide groups against both an independent peak picking algorithm and the three-dimensional crystal structure of azurin, and a coincidence of about 95% is observed. Automated 'demixing' of components that are 'mixed' in a complex manner due to overlap of the HN and/or (15)N frequencies is illustrated.

Azurin↗