Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin.

Abstract

Abstract. Antibodies with epitopes near the heavy meromyosin/light meromyosin junction distinguish the folded from the extended conformational states of smooth muscle myosin. Antibody 10S.1 has 100-fold higher avidity for folded than for extended myosin, while antibody S2.2 binds preferentially to the extended state. The properties of these antibodies provide direct evidence that the conformation of the rod is different in the folded than the extended monomeric state, and suggest that this perturbation may extend into the subfragment 2 region of the rod. Two antihead antibodies with epitopes on the heavy chain map at or near the head/rod junction. Magnesium greatly enhances the binding of these antibodies to myosin, showing that the conformation of the heavy chain in the neck region changes upon divalent cation binding to the regulatory light chain. Myosin assembly is also altered by antibody binding. Antibodies that bind to the central region of the rod block disassembly of iliamerits upon MgATP addition. Antibodies with epitopes near the COOH terminus of the rod, in contrast, promote filament depolymerization, suggesting that this region of the tail is important for assembly. The monoctonal antibodies described here are therefore useful both for detecting and altering conformational states of smooth muscle myosin.

Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures

“…The specificity of antibody recognition has proven to be a useful tool for detecting and mapping the tertiary structure of many proteins, including, for example, myosin (32,81,89), creatine kinase (59), p53 (20,28,93), influenza RNA polymerase (79), and hepatitis C virus E2 glycoprotein (14,80). In this study, we found that several antibodies specific for the HSV ICP8 SSB can serve as immunochemical probes for the investigation of ICP8 topology during replication complex assembly.…”

Myosin filament assembly in an ever-changing myofilament lattice of smooth muscle

“…The required critical concentration increases dramatically when ATP is present in the solution (32) and decreases when the regulatory light chain of the myosin is phosphorylated (32,66). A short region near the carboxy terminus (tail end) of myosin has been shown to be crucial for filament self-assembly (33,63). Cross et al (9) proposed that a tail tip-to-tail tip association of two myosin molecules is responsible for forming an anti-parallel dimer, which then serves as a nucleation site for filament growth.…”

The Two Heads of Smooth Muscle Myosin Are Enzymatically Independent but Mechanically Interactive

“…Polyacrylamide SDS gels run on these supernatants confirmed the accuracy of the concentrations and indicated that the amount of contaminating actin was less than 5%. HMMs were anchored to the coverslip using antibody S2.2 (18), and actin filament movement was measured as described previously (16,19). The basic buffer typically used for actin treatment and washing in the flow cell (see below) contained 60 mM KCl, 25 mM imidazole-HCl (pH 7.4 at 30°C), 4 mM MgCl 2 , 1 mM EGTA, and 10 mM DTT (buffer A).…”