Browsing by Author "Lemmon, Christopher A"

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    Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants.
    (Biochemistry, 2017-08-11) Ohashi, Tomoo; Lemmon, Christopher A; Erickson, Harold P
    To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔ(I)1-5, FNΔ(III)1-3, FNΔ(III)4-8, and FNΔ(III)11-14. A monomeric form, FNmono, which lacked the C-terminal dimerization region, was also created. FNtnA-D was generated by swapping FNIII domains 1-8 in FNΔ(III)11-14 with seven FNIII domains from tenascin-C. The conformations of these mutants were analyzed by glycerol gradient sedimentation under low-salt (20 mM NaCl) and high-salt (200 mM NaCl) conditions. Surprisingly, most of the mutants showed a compact conformation under low-salt conditions, except for FNtnA-D. When we tested these mutants in cell culture, FNΔ(I)1-5, FNΔ(III)1-3, and FNtnA-D were unable to form a matrix. Interestingly, FNΔ(III)1-3 and FNtnA-D were capable of co-assembly with full-length FN, while FNΔ(I)1-5 was not. This indicates that the segment (I)1-5 is crucial for matrix assembly and segment (III)1-3 is also important. Mutations in FN are associated with glomerulopathy, but when we studied mutant proteins, the single-nucleotide mutations had only minor effects on conformation and matrix assembly. The mutations may destabilize their FNIII domains or generate dimers of dimers by disulfide cross-linking.
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    Probing the folded state of fibronectin type III domains in stretched fibrils by measuring buried cysteine accessibility.
    (The Journal of biological chemistry, 2011-07) Lemmon, Christopher A; Ohashi, Tomoo; Erickson, Harold P
    Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.