Vitali Tugarinov

Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy.

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

Methyl Groups as Probes of Structure and Dynamics in NMR Studies of High-Molecular-Weight Proteins

Methyl groups are of particular interest in NMR studies of proteins since they occur frequently in the hydrophobic cores of these molecules and thus are often sensitive reporters of structure and dynamics. Methyl probes can play a very important role in applications that involve high-molecular-weight proteins because of favorable properties that facilitate the recording of NMR spectra with high sensitivity and resolution. First, the threefold degeneracy of methyl protons in CH3 isotopomers (CH3, CH2D, and CHD2 methyls will be considered in this review) effectively increases the concentration of each group significantly beyond that for, say, backbone amides. Second, because methyl groups are localized at the peripheries of side chains, many tend to be dynamic; this leads to slower relaxation that can be exploited in studies of large systems. Third, in the past few years it has become possible to produce proteins in which methyl groups are selectively protonated in a highly deuterated background; this leads to further enhanced relaxation properties that greatly increase the size of systems that can be studied. Fourth, distances between proximal methyl groups, established on the basis of NOEs, often connect regions of the molecule that are far removed in primary structure. In addition, these moieties serve as probes in investigations of protein–ligand interactions, 10] fast and slow timescale side-chain dynamics, dynamics of protein folding, and in the detection of proteins and complexes in in-cell NMR experiments. In this Minireview, we focus on using methyl groups to study both structure and dynamics in high-molecular-weight proteins. A key aspect has been the interplay between new isotope-labeling methodology and NMR techniques that are specifically designed for a given labeling pattern. Thus, a description of the new labeling approaches is first presented, followed by a brief summary of the NMR experiments that have been developed for site-specific methyl assignments. The relaxation properties of methyl groups are discussed, and basic principles of methyl-TROSY spectroscopy are presented. Finally, a number of practical applications involving global protein-fold determination and studies of side-chain dynamics are described. The approaches and concepts described here are illustrated with applications to the enzyme malate synthase G (MSG) from E. coli—a monomeric 723-residue protein (82 kDa) that has been extensively characterized by NMR in our laboratory over the past several years and whose global fold has been recently derived de novo from NMR data exclusively. MSG is a four-domain enzyme that catalyzes the Claisen condensation of glyoxylate and acetyl-CoA to produce malate and is a part of a biosynthetic bypass (“glyoxylate shunt”) that is activated in many pathogenic microorganisms under anaerobic conditions. Since the glyoxylate shunt is absent in man, the enzymes of this bypass have recently been recognized as potential targets for drug design to improve existing antibiotic agents.

Quantitative NMR Studies of High Molecular Weight Proteins: Application to Domain Orientation and Ligand Binding in the 723 Residue Enzyme Malate Synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. 15N-1HN residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of 1HN, 15N and 13CO nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glyoxalate binding, approximately 1.2 x 10(6)M(-1)s(-1) and approximately 2.7 x 10(6)M(-1)s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed.

NMR Structure of an Anti-Gp120 Antibody Complex with a V3 Peptide Reveals a Surface Important for Co-Receptor Binding

BACKGROUND The protein 0.5beta is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1(IIIB) strain. The epitope recognized by 0.5beta is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. RESULTS Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5beta in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5beta Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5beta encompasses 14 of the 18 P1053 residues. The bound peptide assumes a beta-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A(2), respectively, in the 0.5beta Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. CONCLUSIONS The surface of P1053 that is in contact with the anti-protein antibody 0.5beta is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5beta antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the beta-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.

A cis proline turn linking two beta-hairpin strands in the solution structure of an antibody-bound HIV-1IIIB V3 peptide.

The refined solution structure of an 18-residue HIV-1IIIB V3 peptide in complex with the Fv fragment of an anti-gp120 antibody reveals an unexpected type VI β-turn comprising residues RGPG at the center of a β-hairpin. The central glycine and proline of this turn are linked by a cis peptide bond. The residues of the turn interact extensively with the antibody Fv. 15N{1H} NOE measurements show that the backbone of the peptide, including the central QRGPGR loop, is well ordered in the complex. The solution structure is significantly different from the X-ray structures of HIV-1MN V3 peptides bound to anti-peptide antibodies. These differences could be due to a two-residue (QR) insertion preceding the GPGR sequence in the HIV-1IIIB strain, and the much longer peptide epitope immobilized by the anti-gp120 antibody.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

Significance Chaperones are integral components of the cellular machinery that assist protein folding and protect against misfolding and aggregation. A bottleneck in understanding how chaperones work is that the relevant functional states are too sparsely populated and dynamic to be observed using conventional biophysical methods. NMR is uniquely suited to detect and provide atomic resolution functional information on such “invisible” states. Here we quantitate the kinetics of the chaperone GroEL binding to a protein substrate that exists in a metastable equilibrium between the native state and a sparsely populated folding intermediate, under conditions where the GroEL-bound states are not directly observable. We show that in the absence of cofactors, GroEL possesses substantial intrinsic un/foldase activity that is mediated by hydrophobic interactions. The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr–Purcell–Meinboom–Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the “invisible” GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from 15N, 1HN, and 13Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.

Deuterium Spin Probes of Backbone Order in Proteins: 2H NMR Relaxation Study of Deuterated Carbon α Sites

(2)H spin relaxation NMR experiments to study the dynamics of deuterated backbone alpha-positions, D(alpha), are developed. To date, solution-state (2)H relaxation measurements in proteins have been confined to side-chain deuterons-primarily (13)CH(2)D or (13)CHD(2) methyl groups. It is shown that quantification of (2)H relaxation rates at D(alpha) backbone positions and the derivation of associated order parameters of C(alpha)-D(alpha) bond vector motions in small [U-(15)N,(13)C,(2)H]-labeled proteins is feasible with reasonable accuracy. The utility of the developed methodology is demonstrated on a pair of proteins-ubiquitin (8.5 kDa) at 10, 27, and 40 degrees C, and a variant of GB1 (6.5 kDa) at 22 degrees C. In both proteins, the D(alpha)-derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from (15)N relaxation rates. Semiquantitative solution-state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for D(alpha) sites in proteins equal to 174 kHz. Using a uniform value of QCC for all D(alpha) sites, we show that C(alpha)-D(alpha) bond vectors are motionally distinct from the backbone amide N-H bond vectors, with (2)H-derived squared order parameters of C(alpha)-D(alpha) bond vector motions, S(2)(CalphaDalpha), on average slightly higher than their N-H amides counterparts, S(2)(NH). For ubiquitin, the (2)H-derived backbone mobility compares well with that found in a 1-mus molecular dynamics simulation.

Methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl groups in large proteins

A set of sensitive methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl positions in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine β and isoleucine γ2 1H–13C methyl chemical shift assignments could be obtained from the set of new methyl-detected ‘out-and-back’ 3D experiments. The described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular weight range irrespective of the labeling strategy chosen to produce selectively protonated Alaβ and Ileγ2 13CH3 sites on a deuterated background.

An NMR Experiment for Simultaneous TROSY-Based Detection of Amide and Methyl Groups in Large Proteins

A sensitive 2D NMR experiment for simultaneous time-shared TROSY-type detection of amide and methyl groups in high-molecular-weight proteins is described. The pulse scheme is designed to preserve the slowly decaying components of both 1H-15N and methyl 13CH3 spin systems in the course of indirect evolution and acquisition periods. The proposed methodology is applied to the study of substrate binding to {U-[15N,2H]; Ile-[13CH3]; Leu,Val-[13CH3/12CD3]}-labeled 82-kDa enzyme Malate Synthase G and is expected to accelerate NMR-based screening of large proteins labeled with 15N and selectively labeled with 13CH3 at methyl sites.

Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy

The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface of negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis of exchange lifetime broadening arising from binding to nanoparticles of differing size. The relative contributions of residence time and rotational tumbling to the total effective correlation time of the bound protein are modulated by nanoparticle size, thereby permitting the various motional and exchange parameters to be determined. The residence time of ubiquitin bound to the surface of both large and small unilamellar liposomes is ∼20 μs. Bound ubiquitin undergoes internal rotation about an axis approximately perpendicular to the lipid surface on a low microsecond time scale (∼2 μs), while simultaneously wobbling in a cone of semiangle 30-55° centered about the internal rotation axis on the nanosecond time scale. The binding interface of ubiquitin with liposomes is mapped by intermolecular paramagnetic relaxation enhancement using Gd(3+)-tagged vesicles, to a predominantly positively charged surface orthogonal to the internal rotation axis.