ABSTRACT
Traditional approaches for macromolecular structure elucidation, including NMR, crystallography and cryo-EM have made significant progress in defining the structures of protein-protein complexes. A substantial number of macromolecular structures, however, have not been examined with atomic detail due to sample size and heterogeneity, or resolution limitations of the technique; therefore, the general applicability of each method is greatly reduced. Synchrotron footprinting attempts to bridge the gap in these methods by monitoring changes in accessible surface areas of discrete macromolecular moieties. As evidenced by our previous studies on RNA folding and DNA-protein interactions, the three-dimensional structure is probed by examining the reactions of these moieties with hydroxyl radicals generated by synchrotron X-rays. Here we report the application of synchrotron footprinting to the investigation of protein- protein interactions, as the novel technique has been utilized to successfully map the contact sites of gelsolin segment-1 in the gelsolin segment 1/actin complex. Footprinting results demonstrate that phenylalanine 104, located on the actin binding helix of gelsolin segment 1, is protected from hydroxyl radical modification in the presence of actin. This change in reactivity results from the specific protection of gelsolin segment-1, consistent with the substantial decrease in solvent accessibility of F104 upon actin binding, as calculated from the crystal structural of the gelsolin segment 1/actin complex. The results presented here establish synchrotron footprinting as a broadly applicable method to probe structural features of macromolecular complexes that are not amenable to conventional approaches.
Subject(s)
Protein Footprinting , Proteins/chemistry , Proteins/metabolism , Synchrotrons , Actins/chemistry , Actins/metabolism , Amino Acid Sequence , Animals , Aspartic Acid/chemistry , Binding Sites , Calcium/chemistry , Gelsolin/chemistry , Gelsolin/metabolism , Hydroxyl Radical/chemistry , Kinetics , Macromolecular Substances , Models, Molecular , Phenylalanine/chemistry , Protein Conformation , Protein Structure, Tertiary , Rabbits , X-RaysABSTRACT
We have crystallized the N-terminal actin binding domain (ABD1) of human fimbrin, a representative member of the largest class of actin crosslinking proteins. Diffraction from these crystals is consistent with the orthorhombic space group P2(1)2(1)2(1) (a = 50.03 A, b = 61.24 A, c = 102.30 A). These crystals contain one molecule in the asymmetric unit and diffract to at least 1.9 A resolution. The crystal structure of ABD1 will be the first structure of an actin crosslinking domain.
