NMR Structural Biology, Protein Biochemistry

vcopiechemistry.montana.edu

Our laboratory specializes in nuclear magnetic resonance (NMR)-based structural biology research. We are particularly interested in understanding the crucial links between the molecular structures, internal dynamics, and biochemical functions of proteins that are of importance to human endeavors. Questions of interest are: What is the connection between a protein's three-dimensional architecture, flexibility of its amino acids and of its structural elements, and its biological function(s)? How do atomic structures and internal dynamics modulate the biochemical activity of proteins? What is the significance of conserved amino acid residues in protein families? Our approach to providing answers to these scientific issues is to use modern multidimensional (2D, 3D, 4D), heteronuclear (1H, 15N, 13C, 2H) solution nuclear magnetic resonance (NMR) spectroscopy in conjunction with complementary biophysical techniques. We are currently investigating the structural and functional properties of several intriguing proteins

This project involves the NMR dynamical studies of functionally altered variants of the tryptophan repressor protein, a member of a large family of proteins that regulate the expression of metabolic genes by binding to DNA operator regions (see Figure). This research is in collaboration with Dr. Jannette Carey, Professor of Biochemistry at Princeton University. Our interest centers on understanding the role(s) played by molecular flexibity (i.e. internal motions of atoms) to modulate the functional properties of the protein. Functional residues of proteins are often located on solvent accessible surface loops positioned within a more stable protein core. The biological activity of a protein is thus intrinsically linked to molecular flexibility. The issues of dynamics and molecular flexibility have been particularly important when trying to understand the fundamental principles underlying molecular recognition, protein-protein and protein-nucleic acid interactions. To this effect, our research focuses on the issue of molecular recognition by bacterial DNA-binding proteins, and in particular, on understanding the dynamical properties of a temperature-sensitive mutant of the tryptophan repressor protein (L75F-TrpR) whose biological and biophysical properties cannot be simply explained by small structural changes when compared to the wild-type repressor protein (WT-TrpR).

Selected Publications

Robert Tyler, Istvan Pelczer, Jannette Carey, and Valerié Copié:
Three-dimensional solution structure of apo-L75F, a temperature-sensitive mutant of the tryptophan repressor protein
Biochemistry, Vol. 41, pp. 11954-11962 (2002)

Keywords:
Biophysical, NMR, Protein Chemistry, Spectroscopy, Structure

We have also become very interested in proteins originating from organisms living in the hot and acidic thermal pool of Yellowstone National Park. This research is part of MSU's Thermobiology Institute (TBI). A research theme that is particularly attractive to us is: What are the characteristic molecular features of proteins originating from thermophilic organisms that distinguish them from their mesophilic partners?
For example, how do organisms adapt to the high temperature (T > 70 oC) , acidic (pH < 4.0), and a toxic metal-rich (arsenic, iron, copper, mercury, and others) environment of YNP thermal pools? How do proteins originating from extremophiles get modified to function in such an environmental context? What changes take place at the molecular levels? How do proteins modify their thermodynamic properties (i.e. thermal stability, flexibility of functional residues) to operate efficiently in thermophilic conditions? How do proteins cope with arsenic, cadmium, or copper-rich environments? Modern multidimensional, heteronuclear (1H, 15N, 13C, 2H) solution nuclear magnetic resonance (NMR) spectroscopy is an excellent technique to provide answers to these fundamental issues, both at the structural and motional levels of atoms, and complements well the X-ray-based structural biology research programs taking place within TBI.

Selected Publications

Lara Taubner, Michelle McGuirl, David Dooley, and Valerié Copié:
¹H, ¹³C, ¹⁵N backbone and sidechain resonance assignments for the 18 kDa apo-NosL, a novel copper(I) containing protein from Achromobacter Cycloclastes
A. Letter to the Editor: Journal of Biomolecular NMR. Vol 29, pp. 211-212 (2004)

Keywords:
NMR, Protein Chemistry, Structure

This research is concerned with understanding the biochemical mechanisms of polydnaviral-mediated suppression of insect immune defenses and insect growth. We propose to undertake detailed structure-function and molecular studies of two cys-motif polydnaviral proteins, VHv1.1 and VHv1.4, aimed at identifying and understanding the basic molecular mechanisms of insect-parasite interactions. The goal of the research is to provide fundamental biochemical insights into insect parasitism and insect control that could be used to enhance plant resistance against insect pests. Polydnaviral cysteine-rich proteins are of interest because they are ubiquitous and interfere with insect functions, acting both as anti-immune and anti-growth agents. The goal of the proposed research is to aid in the design of synthetic mimics based on cys-motif protein structures and protein chemistry, and in the engineering of transgenic crops encoding insect resistance genes. To this end, we have defined three specific aims to determine the high-resolution 3D structures and to identify the functional determinants of polydnaviral cysteine-rich proteins mediating their functions in insect hosts following endoparasitic wasp oviposition. This research is in collaboration with Dr. Bruce A. Webb, Professor of Entomology at the University of Kentucky, and an expert in endoparasitic wasp and polydnavirus biology.

Selected Publications

Taubner LM, McGuirl MA, Dooley DM, and Copié V:
Structural studies of Apo NosL, an accessory protein of the nitrous oxide reductase system: insights from structural homology with MerB, a mercury resistance protein
Biochemistry vol. 45 pp.12240-52 (2006)

Jerrod Einerwold, Mahesh Jaseja, Kenneth Hapner, Bruce Webb, and Valerié Copié:
Solution structure of the carboxyl terminal cysteine rich domain of the VHv1.1 polydnaviral gene product: comparison with other cystine knot structural folds.
Biochemistry, Vol 40, pp 14404-14412 (2001)

Lara Taubner, Michelle McGuirl, David Dooley, and Valerié Copié:
Three-dimensional structure of the apo-form of the copper(I) binding protein NosL, originating from the nos gene cluster of Achromobacter Cycloclastes.
B. Biochemistry, Manuscript in preparation, (2006)

Keywords:
Biochemistry, Protein Chemistry, Structure

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