Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Probing the Binding Sites of the Actin/Cofilin Complex By Synchrotron Protein Footprinting

The assembly of biological macromolecules into functional complexes is directed by specific interactions that occur between the partners. The elucidation of the functional interface facilitates an extensive understanding of the critical biological processes these molecules modulate. Traditional structural approaches, such as mutagenesis, NMR spectroscopy, and X-ray crystallography afford descriptions of such macromolecular complexes. Synchrotron protein footprinting is a new method that monitors changes in accessible surface areas of discrete sites of a macromolecule by investigating their relative reactivity to hydroxyl radicals generated by synchrotron X-rays. The millisecond resolution afforded by synchrotron protein footprinting allows for kinetic investigations into biological processes, as disclosed by the determination of the rate constants for RNA folding [1-3]. Our extension of these footprinting studies to proteins has been previously documented [4-5]. Here we report the first direct determination of a previously unknown interface site of cofilin bound to the G-actin monomer.

Cofilin binds actin monomers and filaments and has a pH dependent actin severing ability. Genetical & biochemical data suggest that the N-terminal segments b1 and a1 and helix a3 provide an "actin binding face" for cofilin. Our footprinting results confirm that b1 and a1 contributed to the binding domains. In the helix a3 (ranging from 95 to 111), while residues 106-117 contributed potentially to the binding sites, those 91-105 were suggested by our footprinting data to significantly contribute the binding sites. Our research also strongly suggested that proline 94, an amino acid between reverse turn 1 and a3, which has not been found to be one binding site, is part of the binding interface. Furthermore, we demonstrated that phenylalanine 124 of a loop ranging from 122 to 129 between b6 and a4, is not protected upon binding. The binding interface of actin is also footprinted, which suggests that the conformation of G-actin changes upon binding cofilin.


  1. Sclavi, B., Chance, M., Brenowitz, M. & Woodson, S. Visualizing RNA Folding at Millisecond Intervals by Synchrotron Hydroxyl Radical Footprinting. Science 279,1940-1943 (1998).
  2. Ralston, C.Y., He, Q., Brenowitz, M. & Chance, M.R. Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation, Nat. Struct. Biol. 7, 371-374 (2000).
  3. Ralston, C.Y., Sclavi, B., Sullivan, M., Deras, M.L., Woodson, S.A., Chance, M.R., Brenowitz, M. Time-resolved synchrotron X-ray footprinting and its application to RNA folding, Meth. Enzymol. 317, 353-368 (2000).
  4. Maleknia, S.M., Brenowitz, M., Chance, M.R. Millisecond Radiolytic Modification of Peptides by Synchrotron X-rays Identified by Mass Spectrometry. Anal. Chem. 71, 3965-3973 (1999).
  5. Maleknia, S.M., Ralston, C. Y., Brenowitz, M. D., Doward, K. M., Chance, M. R. Determination of Macromolecular Folding and Structure by Synchrotron X-Ray Radiolysis Techniques. Anal. Biochem. 2001, in press.

Jing-Qu Guan, Mark R. Chance and Steven C. Almo

Synchrotron Center For Biosciences, Albert Einstein College of Medicine, Ullmann 315, 1300 Morris Park Avenue, New York 10461
*Phone: 718 430 2894; E-mail: jguan@aecom.yu.edu