Exposure of thiol groups and bound nucleotide in G-actin: thiols as an indicator for the native state of actin

Abstract

In monomeric actin the number of thiol groups exposed to thiol reagents and the nucleotide bound are found to be correlated. G-actin, prepared as normally in the presence of ATP, exposed one thiol group (nSH = 1). In the presence of 1 equivalent ADP, as found associated with G-actin preparations when no nucleotide is added, the protein exposed four thiol groups (nSH = 4). When G-actin was prepared in a high excess of ADP (50 eq.) two thiol groups became exposed (nSH = 2). Actin also exposed four thiol groups when depolymerized in buffers containing 10 eq. APCPP or APPCP, with a time course of the thiol-titration similar to that obtained when the protein was prepared in a nucleotide-free buffer. When actin was depolymerized in a buffer containing 10 eq. APPNP it also exposed 4 thiols; however, titration kinetics are different. In this case, one thiol group reacted quickly, while the reaction of the three others was retarded. Finally, when actin was depolymerized in the ADP-analog APCP it also exposed four thiol groups, with titration kinetics similar to those obtained for actin in nucleotide free buffer. It was concluded that addition of ATP induced a shielding effect on three out of four thiol groups in monomeric actin. ADP (50 eq.) shielded two of the four thiol groups, while ATP- and ADP-analogs had no shielding effect. The thiol shielding activity and the protective capacity of a nucleotide are interrelated. Actin preparations, in ATP or ADP (high excess) containing buffers, with one or two thiol groups exposed respectively, are stable and polymerizable over many hours. Actin prepared in buffers containing ATP- or ADP-analogs, exposing four thiol groups, is denatured, losing its capacity to polymerize within few hours. Finally, actin preparations in nucleotide free buffers, with four thiol groups exposed, are rapidly denatured, losing the capacity to polymerize within less than one hour. Thus denaturation of monomeric actin must be understood in terms of loss of thiol shielding. In actin preparations generally the ability to polymerize was lost when, even after addition of ATP to solutions of G-actin, the number of thiol groups exposed was greater than two. It was concluded that in monomeric actin changes in the accessibility of thiol groups, loss of nucleotide binding capacity as well as loss of the polymerization capability of the protein are events which probably represent different aspects of the denaturation process in G-actin.(ABSTRACT TRUNCATED AT 400 WORDS)