The accessibility of etheno‐nucleotides to collisional quenchers and the nucleotide cleft in G‐ and F‐actin

Abstract

Recent publication of the atomic structure of G‐actin (Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes, K.C., 1990, Nature 347, 37–44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno‐nucleotides bound to G‐ and F‐actin were examined in order to assess polymerization‐related changes in the nucleotide phosphate site. The Mg2+‐induced polymerization of actin quenched the fluorescence of the etheno‐nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F‐actin than G‐actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G‐ and F‐actin was greatest for I−, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization‐related movements of charged residues in the atomic structure of G‐actin are suggested. The binding of S‐1 to ϵ‐ATP‐G‐actin increased the accessibility of the etheno group to I− even over that in Mg2+‐polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S‐1 to actin. Thus, the binding of S‐1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin. The pH dependence of collisional quenching shows that the cleft region is more accessible to collisional quenchers at pH 7 than at higher pH and suggests that changes in the environment of the cleft might contribute to the faster polymerization rates observed at lower pH. Copyright © 1992 The Protein Society