pH-induced changes in G-actin conformation and metal affinity.
Metal-induced conformational changes in actin at 20 degrees C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+ concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+ concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+ binding to specific sites of moderate affinity [Zimmerle, C.T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545-6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+ addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+ concentrations (less than 200 microM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+ addition to Ca2+-actin are not associated with changes in the Mg2+ affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+ binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Zimmerle
C T
CT
Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Frieden
C
C
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D008274:Magnesium; D002118:Calcium