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Olga Gursky, Ph.D.Associate Professor of Physiology and Biophysics M.S., Moscow State University
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ResearchProtein Folding, Structure and StabilityAnalysis of the energetic-structure-function relationship and folding pathways in proteins and peptides by circular dichroism spectroscopy, differential scanning calorimetry, fluorescence, x-ray crystallography, and site-directed mutagenesis. The on-going NIH-funded work is aimed at understanding the energetic and structural basis for the conformational plasticity of apolipoproteins. These are protein constituents of lipoproteins that mediate lipid transport and metabolism and are central in the pathogenesis of atherosclerosis, stroke, and certain forms of amyloidosis. Apolipoproteins are distinct in their structural adaptability to various lipoprotein particles and to plasma. We try to understand in molecular detail the energetic and structural basis for this adaptability. A major focus of our research is on the molecular mechanisms of lipoprotein stabilization and fusion. In 2002 we revealed that lipoprotein stability is determined by kinetic barriers. Similar barriers may modulate in-vivo lipoprotein transformations. Our goal is to obtain key molecular determinants for these energy barriers.
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Overview of Earlier Work on Plasma Apolipoproteins
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On-going ProjectsStructural and thermodynamic analysis of human apolipoprotein C-1
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Early folding intermediates of Alzheimer's amyloid A-beta(1-40) peptide.Such folding intermediates are implicated to be pathogenic and may provide potential therapeutic targets against Alzheimer's disease. We used CD spectroscopy and gel electrophoresis to correlate the effects of temperature and peptide concentration on the peptide conformation in water. We detected a rapid reversible heat-induced coil to β-strand transition in Aβ(1-40) that is independent of the peptide concentration and thus is not coupled to aggregation. This is the first observation of the β-strand folding in Aβ that may occur in the Aβ(1-40) monomer or dimer. Our results demonstrate the importance of temperature and thermal history for the Aβ conformation in aqueous solution. |
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Molecular Mechanism of Lipoprotein Stabilization
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Selected PublicationsJayaraman S., Gantz D.L., and Gursky O. 2006. Effects of salt on thermal stability of human plasma high-density lipoproteins. Biochemistry 45: 4620-4628. Benjwal S., Verma S., Röhm K. H., and Gursky O. 2006. Monitoring protein aggregation during thermal unfolding in circular dichroism experiments Protein Science 15: 635-639. Benjwal S., Jayaraman S. and Gursky O. 2005. Electrostatic effects on the kinetic stability of model discoidal high-density lipoproteins. Biochemistry 44:10218-10226. Gursky O. 2005. Apolipoprotein structure and dynamics. Curr. Opin. Lipidol. 16(3): 287-294. Chung C. M., Chiu J. D., Connors L. H., Gursky O., Lim A., Dykstra A. B., Liepnieks J., Benson M.D., Costello C. E., Skinner M., and Walsh M. T. 2005. Thermodynamic Stability of a kI Immunoglobulin Light Chain: Relevance to Multiple Myeloma. Biophys. J. 88: 4232-4242. Jayaraman S., Gantz D.L., Gursky O. 2005. Kinetic stabilization and fusion of discoidal lipoproteins containing human apoA-2 and DMPC: Comparison with apoA-1 and apoC-1. Biophys. J. 88: 2907-2918. Jayaraman S., Gantz D.L., and Gursky O. 2005. Structural basis for thermal stability of human low-density lipoprotein. Biochemistry 44(10): 3965-3971. Jayaraman S., Gantz D.L., and Gursky O. 2004. Poly(ethylene glycol)-induced fusion and destabilization of human high-density lipoproteins. Biochemistry 43: 5520-5531. Ranjana Mehta, Gantz D. L., and Gursky O. 2003. Human plasma high-density lipoproteins are stabilized by kinetic factors J. Mol. Biol. 328(1):183-192 [Abstract] Fang Y., Gursky O., and Atkinson D. 2003. Lipid binding studies of human apolipoprotein A-1 and its terminally truncated mutants. Biochemistry 42 (in press) Ranjana Mehta, Gantz D. L., and Gursky O. 2003. Effects of mutations on the reconstitution and kinetic stability of discoidal lipoproteins. Biochemistry 42: 4751-4758. [Abstract] Fang Y., Gursky O., and Atkinson D. 2003. Effects of the N- and C-terminal deletions on the structure and stability of human apolipoprotein A-1. Biochemistry 42(22): 6881-6890. [Abstract] Gursky O., Ranjana, and Gantz D. L. 2002. Complex of human apolipoprotein C-1 with phospholipid: Thermodynamic or kinetic stability? Biochemistry 41: 7373-7384. [Abstract] Gursky O. 2001. Solution conformation of human apolipoprotein C-1 inferred from Pro mutagenesis: Far- and near-UV CD study. Biochemistry 40: 12178-12185. [Abstract] Gorshkova I. N., Liadaki K., Gursky O., Atkinson D., and Zannis V. I. 2000. Probing the solution structure of apolipoprotein A-1 by mutations, circular dichroism, and fluorescence spectroscopy, Biochemistry 39(51): 15910-19. Gursky O. and Aleshkov S. 2000. Temperature-dependent ß-sheet formation in Alzheimer's amyloid Aß(1-40) peptide: Uncoupling ß-structure folding from aggregation. Biochimica et Biophysica Acta / Protein Structure and Enzymology 1436(1): 93-102. [Abstract] [Full text] Gursky O. 1999. Probing the conformation of human apolipoprotein C-1 by point mutations and trimethyamine-N-oxide. Protein Science 8(10): 2055-2064. [Abstract] Gursky O. and D. Atkinson. 1998. Thermodynamic analysis of human plasma apolipoprotein C-1: High-temperature unfolding and low-temperature oligomer dissociation. Biochemistry 37(5): 1283-1291. [Abstract] Gursky O. and D. Atkinson. 1996. High- and low-temperature unfolding of human high-density apolipoprotein A-2. Protein Science 5 (9): 1874-1882. Gursky O. and D. Atkinson. 1996. Thermal unfolding of human high-density apolipoprotein A-1: Implications for a lipid-free molten globular state. Proc. Natl. Acad. Sci. USA 93: 2991-2995. [Abstract] [Full text] Gursky O., E. Fontano, B. Bhyravbhatla and D. L. D. Caspar. 1994. Stereospecific dihaloalkane binding in a pH-sensitive cavity in cubic insulin crystals. Proc. Natl. Acad. Sci. USA 91: 12388-12392. [Abstract] [Full text] Badger J., A. Kapulsky, O. Gursky, B. Bhyravbhatla, and D. L. D. Caspar. 1994. Structure and selectivity of a monovalent cation binding site in cubic insulin. Biophys. Journal 66: 286-292. Gursky O., J. Badger, Y. Li, and D. L. D. Caspar. 1992. Conformational changes in cubic insulin crystals in the pH range 7-11. Biophysical Journal 63: 1210-1220. [Abstract] Gursky O., Y. Li, J. Badger, and D. L. D. Caspar. 1992. Monovalent cation binding to cubic insulin crystals. Biophysical Journal 61: 604-611. [Abstract] Lvov Yu. M., O. B. Gurskaja, T. S. Berzina, and V. I. Troitsky. 1989. Structure of Langmuir-Blodgett superlattices with alternative bilayers of Barium Behenate, Phtalocyanine, and Octadecylethylenphenole. Thin Solid Films 182: 283-296. Lvov Yu. M. and O. B. Gurskaya. 1989. Investigation of Langmuir-Blodgett films by the methods of X-ray small-angle diffractometry and reflectometry. Sov. Phys. Crystallography 34(5): 749-752. List of citations on PubMed
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The Group
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Contact Us
Department of Physiology and Biophysics
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The CD spectroscopic and calorimetric analyses of the origins of the structural adaptability in human apoA-1, apoA-2, and apoC-1, supported by other studies, showed that lipid-free apolipoproteins have energetic and structural characteristics of compact protein folding intermediates, with substantial α-helical content but lax tertiary structures. We propose that the exchangeable apolipoproteins may represent a new class of proteins that perform their major physiological function, namely lipid binding, via the molten globule-like state. This laxly folded state may also facilitate the amyloid fibril formation by apolipoproteins.
To advance the energetic-structure-function analysis of apolipoproteins to a level of individual amino acids, we use the smallest human apolipoprotein C-1 (6kD). The sequence homology, secondary structural, and functional similarity of apoC-1 and larger apolipoproteins make it an attractive model system. We analyzed solution structure of apolipoprotein C-1 (apoC-1, 6.6 kD) by circular dichroism of its fifteen mutants containing single Pro or Ala substitutions in the predicted α-helical regions. The results suggest that apoC-1 molecule in solution comprises two dynamic helices connected by a short linker containing residues 30-33 and stabilized by interhelical interactions. We propose that the minimal folding unit in this and other exchangeable apolipoproteins comprises helix-turn-helix formed of four 11-mer sequence repeats and stabilized by interhelical interactions. Comparison of the helical content in lipid-free and lipid-bound apoC-1 suggests that lipid binding shifts the conformational equilibrium towards pre-existing highly helical conformation. Remarkably, near-UV CD spectra of wild type and mutant apoC-1 are not significantly altered upon thermal or chemical unfolding, and thus result from residual aromatic clustering. Correlation of far- and near-UV CD of the mutant peptides suggests that the hydrophobic cluster containing W41 is essential for the helical stability and may form helix nucleation site in apoC-1. 
Lipid-protein association increases the helical content in apolipoproteins and reduces their susceptibility to chemical and thermal denaturation and proteolysis, but the mechanism of this stabilization in unclear. Our spectroscopic and electron microscopic studies have shown that, contrary to the widely held assumption, lipoprotein stability is determined by kinetic rather than thermodynamic factors. We demonstrated that high kinetic barriers for lipoprotein denaturation arise from the particle fusion that accompanies apolipoprotein unfolding and thereby compensates for the solvent exposure of the apolar lipid moieties. Based on our kinetic analyses of discoidal and spherical HDL, we propose that the kinetic mechanism provides a universal natural strategy for lipoprotein stabilization that reconciles the requirements for structural stability and heterogeneity of lipoproteins, prevents spontaneous lipoprotein interconversions, and regulates lipoprotein functions and lifetime in plasma. In the on-going work, we use circular dichroism and fluorescence spectroscopy, light scattering, and electron microscopy in conjunction with protein mutagenesis to gain a detailed understanding of the protein and lipid contributions to the kinetic barriers that define lipoprotein stability.
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