Dmitri Klimov, Professor
School of Systems Biology

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Research Interests
BINF690 Numerical Methods in Bioinformatics
BINF740 Introduction to Biophysics
BINF739/BIOL691 Molecular Modeling for Biologists
BINF741 Introduction to Computer Simulations of Biomolecules


Research Interests

My research interests are focused on two areas of computational study of protein aggregation and unfolding. The first is focused on the assembly of Abeta amyloids, which cause Alzheimer's disease. The second involves the computational investigation of forced (mechanical) unfolding of proteins. The proposed research program is based on the all-atom molecular dynamics (MD) simulations of proteins or peptides in explicit solvent. Both topics are highly important for understanding the molecular aspects of Alzheimer's disease and mechanical functions of proteins in living organisms.

Aggregation of Abeta peptides

The purpose of the computational studies of aggregation is to understand, at molecular level, the formation of Abeta amyloid fibrils, which triggers the onset of Alzheimer's disease. It is recognized that the development of effective therapeutic treatments demands thorough understanding of the kinetics of Abeta peptides' aggregation as well as their fibril structures. Recent experiments provided important information on Abeta fibrils. Solid-state NMR studies [1-3] showed that Abeta peptides (including the wild-type Abeta1-40 and the truncated fragment Abeta10-35) form parallel in-registry beta-sheets (a two-dimensional structure). Three-dimensional structural arrangements of Abeta1-40 and Abeta10-35 fibrils have also been proposed based on simple energetic considerations [2,4]. In contrast, short fragments of Abeta peptides (such as Abeta16-22 and Abeta34-42}) form antiparallel in-registry beta-sheets [5,6]. The kinetics of the assembly of Abeta oligomers (precursors to amyloid fibrils) has been probed by Teplow and coworkers [7]. They found that in all eighteen Abeta peptides, which differ in length and residue substitutions, a transient on-pathway alpha-helical intermediate is observed.

Nevertheless, the molecular basis for the experimental results described above remains poorly understood. To address these issues we initiated MD studies of aggregation of fragments of Abeta peptides ([8,9], see also commentary [10]). For the first time the formation of Abeta oligomers was observed in silica, starting with random initial conditions. In accord with experimental data, MD simulations showed that antiparallel orientation of Abeta16-22 peptides is energetically preferred. Dramatic conformational changes due to interpeptide interactions were observed, which include obligatory alpha-helical intermediate. We established that oligomerization of Abeta peptides is initially driven by hydrophobic interactions. However, electrostatic interactions confer antiparallel registry of peptides, which results in further decrease in potential energy. This kinetic mechanism of Abeta oligomerization may represent a universal pathway of amyloid fibril formation.

Force-induced unfolding of proteins

Force-induced (mechanical) unfolding of proteins is implicated in muscle contraction and relaxation [11], cell adhesion and receptor binding [12], tissue elasticity [11] etc. For example, titin molecule provides passive elasticity for muscle tissues and is composed of about 300 folded domains, among which are immunoglobulin-type (Ig) and fibronectin-type (Fn) domains. Modular construction transforms titin into a complex nonlinear spring [13]. Another mechanically active protein, fibronectin, is a part of extracellular matrix (ECM), which determines elasticity and tensile strength of tissues [11]. Similar to titin, fibronectin includes several types of domains, some of which (Fn3 domains) incorporate binding sites for integrin and heparin molecules [14-16]. It is likely that stretching of Fn3 domains changes the affinity of binding regions and also exposes new "cryptic" association sites [17-18]. Therefore, mechanical unfolding of fibronectin controls the assembly of ECM amyloid fibrils, cell adhesion and migration [12].

Recent atomic force microscopy (AFM) experiments [19] measured, at single molecule level, the unfolding forces of various domains in mechanically active proteins, including titin and fibronectin. The AFM experiments revealed hierarchic (``one by one'') unfolding of domains in the proteins, which starts with the weakest one. Interestingly, the stability of protein domains against thermal or chemical denaturation does not correlate with the mechanical stability [20]. MD simulations provided detailed information on the structural events occurring upon force-induced unfolding of individual domains [21]. Computer simulations performed by us using low-resolution protein models identified the native topology as the key factor determining the unfolding pathways and rapture forces in proteins [22]. In addition, we developed a novel algorithm, which provides accurate prediction of mechanical stability and force-induced unfolding pathways of wild-type proteins [22].

  1. Antzutkin, O.N., Balbach, J.J., Leapman, R.D., Rizzo, N.W., Reed, J., and Tycko, R. (2000) Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of beta-sheets in Alzheimer's beta-amyloid fibrils. Proc. Natl. Acad. Sci. USA 97, 13045-13050.

  2. Petkova, A.T., Ishii, Y., Balbach, J.J., Antzutkin, O.N., Leapman, R.D., Delaglio, F., and Tycko, R. (2002) A structural model for Alzheimer's beta-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. USA 99, 16742-16747.

  3. Burkoth, T.S., Benzinger, T.L.S., Urban, V., Morgan, D.M., Gregory, D.M., Thiyagarajan, P., Botto, R.E., Meredith, S.C., and Lynn, D.G. (2000) Structure of the beta-Amyloid(10-35) fibril. J. Am. Chem. Soc. 122, 7883-7889.

  4. Ma, B. and Nussinov, R. (2002) Stabilities and conformations of Alzheimer's beta-amyloid peptide oligomers (Abeta16-22, Abeta16-35, Abeta10-35): Sequence effects. Proc. Natl. Acad. Sci. USA 99, 14126-14131.

  5. Balbach, J.J., Ishii, Y., Antzutkin, O.N., Leapman, R.D., Rizzo, N.W., Dyda, F., Reed, J., and Tycko, R. (2000) Amyloid fibril formation by Abeta16-22, a seven-residue fragment of the Alzheimer's beta-amyloid peptide, and structural characterization by solid state NMR. Biochem. 39, 13748-13759.

  6. Lansbury, P.T., Costa, P.R., Griffiths, J.M., Simon, E.J., Auger, M., Halverson, K.J., Kocisko, D.A., Hendsch, Z.S., Ashburn, T.T., Spencer, R.G.S. et al. (1995) Structural model for the beta-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide. Nat. Struct. Biol. 2, 990-998.

  7. Kirkitadze, M.D., Condron, M.M., and Teplow, D.B. (2001) Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis. J. Mol. Biol. 312, 1103-1119.

  8. Klimov, D.K. and Thirumalai, D. (2003) Dissecting the assembly of Abeta16-22 amyloid peptides into antiparallel beta-sheets. Structure 11, 295-307.

  9. Lynn, D.G., Snyder, J.P., Lakdawala, A.S., Dong, J., and Lu, K. (2003) Conformational evolution: The wiggling of peptides into amyloid. Structure 11, 242.

  10. Balbirnie, M., Grothe, R., and Eisenberg, D.S. (2001) An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid. Proc. Natl. Acad. Sci. USA 98, 2375-2380.

  11. Massi, F., Klimov, D., Thirumalai, D., and Straub, J. E. (2002) Charged states rather than propensity for beta-structure determine enhanced fibrillogenesis in wild-type Alzheimer's beta-amyloid peptide compared to E22Q Dutch mutant. Prot. Sci. 11, 1639-1647.

  12. Erickson, H.P. (1994) Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin. Proc. Natl. Acad. Sci. USA 91, 10114-10118.

  13. Hynes, R.O. (1999) The dynamic dialogue between cells and matrices: Implications of fibronectin's elasticity. Proc. Natl. Acad. Sci. USA 96, 2588-2590.

  14. Erickson, H.P. (1997) Stretching single protein modules: Titin is a weird spring. Science 276, 1090-1093.

  15. Leahy, D.J., Aukhil, I.L., and Erickson, H.P. (1996) 2.0\AA \ \ crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 84, 155-164.

  16. Johanssen, S., Svineng, G., Wennerberg, K., Armulik, A., and Lohikangas, L. (1997) Fibronectin-integrin interactions. Frontiers Biosci. 2, 126-146.

  17. Sharma, A., Askari, J.A., Humphries, M.J., Jones, E.Y., Stuart, D.I. (1999) Crystal structure of a heparin- and integrin-binding segments of human fibronectin. Eur. Mol. Biol. Org. J. 18, 1468-1479.

  18. Ingham, K.C., Brew, S.A., Huff, S., and Litvinovich, S.V. (1997) Cryptic self-association sites in type III modules of fibronectin. J. Biol. Chem. 272, 1718-1724.

  19. Litvinovich, S.V., Brew, S.A., Aota, S., Akiyama, S.K., Haudenschild, C., and Ingham, K.C. (1998) Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type {III} module. J. Mol. Biol. 280, 245-258.

  20. Fisher, T.E., Oberhauser, A.F., Carrion-Vazquez, M., Marszalek, P.E., and Fernandez, J.M. (1999) The study of protein mechanics with the atomic force microscope. Trends in Biochem. Sci. 24, 379-384.

  21. Li, H., Oberhouser, A.F., Fowler, S.B., Clarke, J., Fernandez, J.M. (2000) Atomic force microscopy reveals the mechanical design of a modular protein. Proc. Natl. Acad. Sci. USA 97, 6527-6531.

  22. Isralewitz, B., Gao, M., and Schulten, K. (2001) Steered molecular dynamics and mechanical functions of proteins. Curr. Opin. Struct. Biol. 11, 224-230.

  23. Klimov, D.K. and Thirumalai, D. (2000) Native topology determines force-induced unfolding pathways in globular proteins. Proc. Natl. Acad. Sci. USA 97, 7254-7259.

  24. Oberhauser, A.F., Badilla-Fernandez, C., Carrion-Vazquez, M., and Fernandez, J.M. (2002) The mechanical hierarchies of fibronectin observed with single-molecule AFM. J. Mol. Biol. 319, 433-447.

  25. Paci, E. and Karplus, M. (1999) Forced unfolding of fibronectin type 3 modules: An analysis by biased molecular dynamics simulations. J. Mol. Biol 288, 441-459.

  26. Litvinovich, S.V. and Ingham, K.C. (1995) Interactions between type III domains in the 110 kDa cell-binding fragment of fibronectin. J. Mol. Biol. 248, 611-626.

  27. Betts, S. & King, J. (1999) There's a right way and a wrong way: in vivo and in vitro folding, misfolding and subunit assembly of the P22 tailspike. Structure 7, R131-R139.