Vitali Tugarinov's NMR research group; University of Maryland, College Park; USA
 

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Link to the lab website.

Research
The research in our laboratory focuses on the studies of high-molecular-weight proteins and supra-molecular complexes in solution by NMR techniques. Solution-based techniques allow the studies of proteins at nearly physiological conditions without need for crystallization. Large proteins and supra-molecular complexes are especially challenging objects for NMR applications because of inherently low sensitivity coupled with high complexity of obtained NMR data. Significant progress in NMR methodology for high-molecular-weight proteins and nucleic acids has been achieved in the last decade and molecular species in the mega-dalton molecular weight range have been analyzed. Important information about protein structure and function as well molecular functional dynamics in solution can be obtained that has so far been inaccessible due to molecular size limitations of NMR applications.
Two principal research directions are followed:

• the development of novel isotope-labeling and NMR methods that allow and facilitate the studies of large proteins and their complexes.
• application of existing and new NMR methodology to high-molecular-weight proteins of significant biological importance (COP9 Signalosome and its components; GroeS co-chaperonine from plants etc.)

Solution studies of structure and dynamics of large and complex protein structures requires preparation of (selectively) isotopically (¹³C, ¹⁵N, ²H) labeled protein samples. Malate Synthase G (MSG, 82-kDa) is used as a model system to test and develop optimal isotope labeling strategies that would be applicable to proteins in the ~100 kDa range.
(a) The x-ray structure (PDB code: 1D8C) and the ten lowest energy NMR structures of MSG calculated on the basis of experimental restraints. Backbone traces of the x-ray structure (left) and NMR structures (right) are displayed and superimposed by aligning residues in elements of regular secondary structure. The α-clasp, α/β, core and C-terminal domains are colored black, green, red and purple, respectively, in the x-ray structure, with the linkers in gray. Individual domains (b: α-clasp, c: α/β, d: core, and e: C-terminal) are displayed and superimposed by fitting over residues in regular secondary structure. The r.m.s deviation of the NMR ensemble (10 structures) and the x-ray are indicated for heavy backbone atoms of regular secondary structure elements for the entire molecule and individual domains. [Tugarinov, V., Choy, W-Y., Orekhov, V.Y. and Kay, L.E. Solution NMR-derived global fold of a monomeric 82-kDa enzyme. (2005) Proc. Natl. Acad. Sci. USA 102, 622]
The majority of proteins involved in fundamental biochemical processes related to metabolism, translation, transcription and protein degradation, exceed 40 kDa in molecular weight. Spectroscopic, isotope-labeling and biochemical methods developed in the last couple of years open new perspectives for the studies of large proteins by solution NMR techniques. This methodology can now help to address interesting biochemical/biophysical problems related to macromolecular structure and functional dynamics of any class of high-molecular-weight systems including integral membrane proteins, chaperones and protein-DNA complexes.
On-going Work
Recently, a sensitive 2D NMR experiment for simultaneous time-shared TROSY-type detection of amide and methyl groups in high-molecular-weight proteins has been developed in my group. 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 the native substrate (glyoxylate) binding to {U-[¹⁵N,²H]; Ile-[¹³CH₃]; Leu,Val-[¹³CH₃/¹²CD₃]} labeled MSG and is expected to accelerate NMR-based screening of large proteins labeled with 15N and selectively labeled with ¹³CH₃ at a number of methyl sites.
(a) Schematicrepresentation of MSG with HN and ILV methyl carbons shown with red and blue circles, respectively. (b) Simultaneous ¹H-¹⁵N/¹³CH₃ correlation map recorded on the 0.5 mM sample of {U-[¹⁵N,²H]; Ile-[¹³CH₃]; Leu,Val-[¹³CH₃/¹²CD₃]}-labeled MSG (37 °C, 600 MHz). Spectral width is adjusted so that all the peaks of isoleucine methyls are aliased. Labeling of F₁ axis with ¹⁵N/¹³C in units of ppm is solely for illustration purposes. (c)-(d) Typical examples of chemical shift changes occurring upon addition of glyoxylate to MSG for (c) the amide peak of W509 and (d) V701 g1 methyl.