Peptide mimetics of an actin-binding site on myosin span two functional domains on actin.
The sites on the myosin heavy chain that interact with actin and are responsible for force generation are ill-defined: crosslinking and experiments with isolated domains of the myosin head implicate regions in both the 50K and 20K (molecular weights in thousands) domains of the myosin head (subfragment 1, S1) in this process. We have synthesized peptides from the sequence around the fast-reacting SH1 thiol residue in the 20K domain of S1 in order to delineate precisely an actin-binding site. We used a combination of 1H-NMR and enzyme inhibition assay and also assessed the effects of peptides on skinned rabbit psoas muscle fibres to show that the region of amino acids 690-725 contains an actin-binding site. Peptides from this region bind to actin, act as mixed inhibitors of the actin-stimulated S1 Mg2(+)-ATPase, and influence the contractile force developed in skinned fibres, whereas peptides flanking this sequence are without effect in our test systems. Remarkably, peptides from the N-terminal half of this segment 690-725 increase force development in skinned fibres at submaximal activating concentrations of Ca2+, that is, they behave as calcium-sensitizers; C-terminal peptides, however, inhibit force development without effecting sensitivity to calcium. These different responses indicate that this region is probably binding at two functionally distinct sites on actin.
Keane
A M
AM
School of Biochemistry, University of Birmingham, UK.
Trayer
I P
IP
Levine
B A
BA
Zeugner
C
C
Ruegg
J C
JC
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009119:Muscle Contraction / Q000187:drug effects; D015879:Myosin Subfragments / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D013438:Sulfhydryl Compounds
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium