In spite of nearly 40 years research on fibronectin (FN), there are two important structural questions about the FN matrix that are unanswered. The first is, what are the bonds that connect FN molecules to make the matrix fibrils? Our first aim is to develop a new approach to identify these FN-FN bonds. We propose to make FN dimers in which 4-6 highly specific 3C and/or TEV protease sites are inserted between certain domains. These FNs will be used to assemble an FN matrix in cell culture. The matrix will be crosslinked to stabilize binding partners, then cleaved with protease. The FN will be released as small fragments, crosslinked to their bonded partners. These will be identified by binding specificity and antibodies. The precise binding sites will be determined by mass spec. The second question is elasticity. An intriguing mechanical feature of FN fibrils is that they can be stretche 4-fold, making them perhaps the most elastic protein polymer known. The mechanism of the elasticity is controversial. One group concludes that stretching involves unfolding and extending FNIII domains. We believe the stretching involves a conformational change of the FN dimers from compact to elongated, without domain unfolding. We propose two approaches to settle the controversy. (1) We have designed FRET constructs using GFP acceptor and chemical fluorophore donors attached to engineered cys in the domain to be tested. The FRET will report the extent of domain unfolding. (2) We will develop shrec, a super-resolution, single-molecule light microscopy technique, to determine the separation of red and green fluorophores in the first FNIII domains in the dimer. Shrec should have a resolution of ~10 nm, and will tell us whether the FN dimers are in the compact conformation (~20 nm apart), extended conformation (~100 nm), or have unraveled FNIII domains (>100 nm). A third project will investigate a rare kidney disease caused by mutations in FN. We believe that the pathology may be caused by a failure to fold into the compact conformation. We propose to test this by examining the FN dimers using established hydrodynamic techniques. We will also apply the shrec technology discussed above to compare the conformations of wild type and mutant FN at the single molecule level.

Public Health Relevance

Fibronectin is the earliest extracellular matrix formed in embryonic tissues and healing wounds. Fibronectin molecules assemble into matrix fibrils, but almost nothing is known about their structure. We propose to determine the molecular structure of protein-protein bonds in matrix fibrils, and the mechanism of fibril elasticity. We will also tet a structural hypothesis for a kidney disease caused by mutations in fibronectin.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA047056-32A1
Application #
8295455
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Ault, Grace S
Project Start
1979-05-01
Project End
2017-03-31
Budget Start
2012-04-13
Budget End
2013-03-31
Support Year
32
Fiscal Year
2012
Total Cost
$311,663
Indirect Cost
$110,642
Name
Duke University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Giacomodonato, Mónica N; Noto Llana, Mariángeles; Aya Castañeda, María Del Rosario et al. (2014) AvrA effector protein of Salmonella enterica serovar Enteritidis is expressed and translocated in mesenteric lymph nodes at late stages of infection in mice. Microbiology 160:1191-9
Ohashi, Tomoo (2014) A fibronectin-derived cell survival peptide belongs to a new class of epiviosamines. J Invest Dermatol 134:882-4
Fouda, Genevieve G; Jaeger, Frederick H; Amos, Joshua D et al. (2013) Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk. Proc Natl Acad Sci U S A 110:18220-5
Lemmon, Christopher A; Ohashi, Tomoo; Erickson, Harold P (2011) Probing the folded state of fibronectin type III domains in stretched fibrils by measuring buried cysteine accessibility. J Biol Chem 286:26375-82
Ohashi, Tomoo; Erickson, Harold P (2011) Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain. J Biol Chem 286:39188-99
Ohashi, Tomoo; Augustus, Anne Marie; Erickson, Harold P (2009) Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin. Biochemistry 48:4189-97
White, Glenn E; Erickson, Harold P (2009) The coiled coils of cohesin are conserved in animals, but not in yeast. PLoS One 4:e4674
Ohashi, Tomoo; Erickson, Harold P (2009) Revisiting the mystery of fibronectin multimers: the fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds. Matrix Biol 28:170-5
Ng, Sean P; Billings, Kate S; Ohashi, Tomoo et al. (2007) Designing an extracellular matrix protein with enhanced mechanical stability. Proc Natl Acad Sci U S A 104:9633-7
Ohashi, Tomoo; Galiacy, Stephane D; Briscoe, Gina et al. (2007) An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci 16:1429-38

Showing the most recent 10 out of 51 publications