Fibronectin is a major protein of blood and tissues that plays a central role in cell adhesion. The goal of GM35719 is to gain a deeper understanding of the three-dimensional relationship of various regions of fibronectin, and of the structural basis for ligand binding and molecular expansion.
The specific aim 1 of this renewal application is to characterize the dynamic interactions between various functional domains of plasma fibronectin using electron spin resonance (ESR) spectroscopy. The motions of the fibronectin molecule and its fragments under various conditions will be studied by estimating the effective rotational correlation times from the experimental spectra using computer simulations.
The specific aim 2 is to determine the intramolecular distances of the fibronectin molecule using fluorescence energy transfer techniques. These distances will be used as new constraints to refine the current three-dimensional model of fibronectin structure.
The specific aim 3 is to study the non-covalent association of fibronectin dimers by using monomeric fibronectin. The contact sites and the nature of non-covalent association between the two monomers of fibronectin, which lack the interchain disulfide bonds, will be studied by using both ESR and fluorescence spectroscopy.
The specific aim 4 is to characterize recombinant fibronectin fragments expressed in bacterial systems using molecular cloning techniques. Site-directed mutagenesis by substitution of a cysteine residue involved in interchain disulfide bonding with a serine residue will be employed to determine whether the two interchain disulfide bridges in the carboxyl ends of the molecule are arranged in parallel or antiparallel fashion.
The specific aim 5 is to investigate the structure and dynamics of the recombinant fibronectin fragments using a combination of site-directed mutagenesis and biophysical techniques. Fibronectin fragments will be genetically engineered to contain single selective labeling sites, one at a time, without affecting the protein function. This permits the placement of spin labels or fluorescent labels into the recombinant fibronectin fragment and the elucidation of detailed structural information through ESR or fluorescence analysis. The proposed studies will provide previously unavailable information regarding the three-dimensional structure of the fibronectin molecule and mechanisms by which functional unmasking of certain domains occurs in response to ligand-induced conformational alterations. This information should provide a basis for explaining important roles of fibronectin in cell attachment and migration, embryogenesis, wound healing, and oncogenic transformation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035719-07
Application #
3288819
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1986-01-01
Project End
1993-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Komarov, A M; Lai, C S (1995) Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy. Biochim Biophys Acta 1272:29-36
Lai, C S; Komarov, A M (1994) Spin trapping of nitric oxide produced in vivo in septic-shock mice. FEBS Lett 345:120-4
Komarov, A M; Joseph, J; Lai, C S (1994) In vivo pharmacokinetics of nitroxides in mice. Biochem Biophys Res Commun 201:1035-42
Lai, C S; Wolff, C E; Novello, D et al. (1993) Solution structure of human plasma fibronectin under different solvent conditions. Fluorescence energy transfer, circular dichroism and light-scattering studies. J Mol Biol 230:625-40
Kar, L; Lai, C S; Wolff, C E et al. (1993) 1H NMR-based determination of the three-dimensional structure of the human plasma fibronectin fragment containing inter-chain disulfide bonds. J Biol Chem 268:8580-9
Komarov, A; Mattson, D; Jones, M M et al. (1993) In vivo spin trapping of nitric oxide in mice. Biochem Biophys Res Commun 195:1191-8
Narasimhan, C; Lai, C S (1991) Differential behavior of the two free sulfhydryl groups of human plasma fibronectin: effects of environmental factors. Biopolymers 31:1159-70
Joseph, J; Shih, C C; Lai, C S (1991) Synthesis of the spin-labeled derivative of an ether-linked phospholipid possessing high antineoplastic activity. Chem Phys Lipids 58:19-26
Squier, T C; Mahaney, J E; Yin, J J et al. (1991) Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy. Biophys J 59:654-69
Wolff, C E; Lai, C S (1990) Inter-sulfhydryl distances in plasma fibronectin determined by fluorescence energy transfer: effect of environmental factors. Biochemistry 29:3354-61

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