Abstract
The gelatin-binding sites of fibronectin are confined to a 42-kDa region having four type I and two type II modules in the following order: I(6)-II(1)-II(2)-I(7)-I(8)-I(9). To determine the relative importance of each module for recognition of gelatin, recombinant green fluorescent fusion proteins were prepared in which individual modules or groups of modules were deleted, and the resulting proteins were tested for binding to gelatin by analytical affinity chromatography. Deletion of both type II modules did not eliminate binding, confirming that at least some of the type I modules in this region are able to bind gelatin. It was found that deletion of type I module 6 tends to increase the affinity, whereas deletion of any other module decreases it. Deletion of module I(9) had a large effect but only if module II(2) was also present, suggesting an interaction between these two noncontiguous modules. Analysis of more than 20 recombinant fusion products led to the conclusion that all modules contribute to the interaction either directly by contacting the ligand or indirectly through module-module interactions.
Highlights
The gelatin-binding sites of fibronectin are confined to a 42-kDa region having four type I and two type II modules in the following order: I6-II1-II2-I7-I8-I9
This region can be isolated as a 42-kDa fragment (42-kDa GBF)1 that binds to gelatin with affinity only slightly lower than that of the parent protein (1)
Since each of the six modules in the 42-kDa gelatin-binding fragment of fibronectin has a unique number, 6, 1, 2, 7, 8, and 9, those numbers are used as a shorthand notation to specify the modular composition of the various mutants, and dashes (-) are used to designate deletions
Summary
Materials—Polyclonal anti-green fluorescent protein (GFP) antibody was purchased from Clontech. The 42-, 30-, and 21-kDa gelatin-binding fragments were purified from a thermolysin digest of human plasma fibronectin as described previously (5). Recombinant GFP-fibronectin fragments were purified on Talon metal affinity resin (Clontech), eluting with 150 mM imidazole, and dialyzed against phosphate-buffered saline (PBS, pH 7.4). The identity of each fragment was verified by detection of the appropriate viral DNA in the infected cells with PCR and by the apparent molecular weight of the purified protein on SDS-PAGE. After washing and blocking with 1% bovine serum albumin in PBS, the plates were incubated with various concentrations of recombinant proteins for 2 h at room temperature. Plates were washed three times with 0.2% bovine serum albumin in PBS and incubated with polyclonal anti-GFP antibody for 1 h at room temperature followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody for 40 min at room temperature. Absorbance was measured with a Dynatec plate reader, and C50% values were obtained by fitting the data to a simple binding isotherm using Sigmaplot
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