Mapping the G-Actin Binding Surface of Cofilin Using Synchrotron Protein Footprinting

Abstract

Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity. Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available. Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex. Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility. The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin. After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117. A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site. Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface. These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin.

Solution Structure of Human Cofilin

“…Site-directed mutagenesis identified Lys 112 and Lys 114 in ␣4 as essential for G-actin binding (40,41). Synchrotron protein footprinting identified residues in yeast cofilin corresponding to Ile 12 , Pro 110 , Met 115 , Ile 124 , and Leu 128 as G-actin binding (42). Of these, Met 115 and Ile 124 correspond to residues showing signal broadening in our HSQC experiments, and Pro 110 and Leu 128 are located adjacent to such signal-broadened residues.…”

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

“…The initial success of this experimental strategy, i.e., combing protein modification by DMBNHS with data dependant multistage tandem mass spectrometry, provides confidence for its application in future studies involving the examination of protein structure in larger proteins, as well as for the study of protein-protein and protein-ligand interactions. C (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) A (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62) D (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) F (97-112) E …”

“…Hydroxyl radical reactions may also lead to cleavage of peptide bonds, cleavage or ring opening of amino acid side chains, or the formation of protein cross-links. Moreover, when the oxidation sites are distributed randomly among several surface amino acid residues, the concentration of each oxidized peptide may be too low to detect [14]. There is also a concern that extensive oxidation may alter a protein's native conformation, thereby leading to erroneous results [15][16][17].…”

Visualizing the Ca 2+ -dependent activation of gelsolin by using synchrotron footprinting

“…Synchrotron X-Ray Radiolysis and MS. Radiolysis experiments were performed at Beamline X-28C of the National Synchrotron Light Source at beam currents ranging between 183 and 195 mA according to published procedures (25)(26)(27). Irradiated protein solutions were subjected to proteolysis by trypsin at an enzymeto-protein ratio of 1:50 at 37°C for 12 h. A Finnigan (San Jose, CA) liquid chromatography quadrupole (LCQ) ion trap mass spectrometer with an electrospray ion source was used to determine the extent of oxidation in the unmodified proteolytic peptides and their radiolytic products.…”

“…Hydroxyl radicals generated from millisecond exposure of aqueous solutions to unattenuated ''white'' synchrotron radiation result in the stable oxidative modification of solvent-accessible amino acid side chains (26). The specific extents and sites of oxidative modification are quantified by proteolytic digestion and MS (25,27). The most reactive residues, which represent the most easily observed experimental probes for this method, include surface-accessible cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, and leucine side chains (26).…”