Structural connectivity in actin: effect of C-terminal modifications on the properties of actin.
In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
R H
RH
Crosbie
C
C
Miller
P
P
Cheung
T
T
Goodnight
A
A
Muhlrad
E
E
Reisler
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D015394:Molecular Structure; D009218:Myosins / Q000378:metabolism; D009282:Naphthalenesulfonates; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship
D000199:Actins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D011108:Polymers; C006421:1,5-I-AEDANS; D009218:Myosins