The intent of this project is to develop new and to improve existing biophysical methodology for the characterization of biological macromolecules in solution, and to apply these methods collaboratively to the study of proteins and their interactions. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. For the study of reversible formation of multi-protein complexes in solution, we have previously developed a novel multi-signal analysis for sedimentation velocity analytical ultracentrifugation. It can permit the label-free detection of sedimentation coefficient distributions of currently up to three protein components, and thus measure the stoichiometry of multiple hydrodynamically separated protein complexes. We have proceeded from the development stage of this method to the collaborative application in several systems. This includes the study of the heterogeneous triple protein complexes discovered for adaptor protein complexes involved in signal transduction after T-cell activation, as well as a study on the ternary interactions of cell surface receptors with HIV envelope protein. We have made significant progress in the theoretical models to account for the influence of reaction kinetics on the sedimentation behavior of interacting systems. This has permitted the more reliable hydrodynamic characterization of proteins by sedimentation velocity analytical ultracentrifugation. This could be applied to collaborative studies of several viral proteins, including the malarial surface protein MSP-3, clathrin basked assembly, the study of the oligomeric state and detergent-binding of detergent-solubilized CB2 receptor, and the interaction of iron-regulatory proteins IRP1 and IRP2 with the iron regulatory elements. With the goal to study conformational heterogeneity and ligand-induced conformational changes of proteins, we have embarked on the adaptation of a difference sedimentation technique to modern Rayleigh interference and absorption optical detection systems. This has promise to increase the sensitivity over existing traditional approaches. We have identified limiting factors in the detection, and developed a protocol for a suitable optical alignment. In the area of optical biosensing, we have further explored the properties of our computational approach to extract information on the functional heterogeneity of surface binding sites from experimental data. This was applied to the characterization of antibody-antigen interactions, and the analysis of integrin receptor with various ligands. In continuation of our studies on small molecule binding to proteins, we have applied surface plasmon resonance (SPR) spectroscopy to characterize new inhibitors of HIV reverse transcriptase and human RNAse H. We have explored the study of protein/ligand stoichiometry by SPR spectroscopy, and the influence of small molecule ligand binding on the protein self-association by sedimentation velocity analytical ultracentrifugation.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Intramural Research (Z01)
Project #
1Z01OD010485-08
Application #
7146054
Study Section
(BEPS)
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Office of the Director, NIH
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Brown, Patrick H; Balbo, Andrea; Schuck, Peter (2007) Using prior knowledge in the determination of macromolecular size-distributions by analytical ultracentrifugation. Biomacromolecules 8:2011-24
Svitel, Juraj; Boukari, Hacene; Van Ryk, Donald et al. (2007) Probing the functional heterogeneity of surface binding sites by analysis of experimental binding traces and the effect of mass transport limitation. Biophys J 92:1742-58
Houtman, Jon C D; Brown, Patrick H; Bowden, Brent et al. (2007) Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling. Protein Sci 16:30-42
Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey et al. (2006) Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus. Proc Natl Acad Sci U S A 103:1882-7
Chen, Zhaochun; Moayeri, Mahtab; Zhou, Yi-Hua et al. (2006) Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. J Infect Dis 193:625-33
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Agniswamy, Johnson; Nagiec, Michal J; Liu, Mengyao et al. (2006) Crystal structure of group A streptococcus Mac-1: insight into dimer-mediated specificity for recognition of human IgG. Structure 14:225-35
Heeb, M J; Schuck, P; Xu, X (2006) Protein S multimers and monomers each have direct anticoagulant activity. J Thromb Haemost 4:385-91
Dam, Julie; Baber, James; Grishaev, Alexander et al. (2006) Variable dimerization of the Ly49A natural killer cell receptor results in differential engagement of its MHC class I ligand. J Mol Biol 362:102-13
Houtman, Jon C D; Yamaguchi, Hiroshi; Barda-Saad, Mira et al. (2006) Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1. Nat Struct Mol Biol 13:798-805

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