Effects of interchain disulfide crosslinks on the trypsin cleavage pattern and conformation of myosin subfragment 2

Abstract

The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the state of the succinimido-ring on the fluorescence and structural properties of pyrene maleimide-labeled alpha alpha-tropomyosin

“…The observation that the pyrene probes at Cys 190, located 2/3 from the amino terminal end, can inhibit polymerizability reinforces the previous result that the binding of pyrene1,-Tm to F-actin is reduced primarily through the weakening of end-to-end interactions (Ishii and Lehrer, 1985). Thus, perturbations can be transmitted over large distances in coiled-coil, rod-like molecules (Edwards and Sykes, 1980;Ueno, 1984;Lu and Lehrer, 1984). It has been postulated that Ca2 -regulation involves troponin perturbation of Tm near Cys 190 (Lehrer et al, 1981), although a model involving direct interaction of troponin at the Tm ends has also been suggested Smillie, 1982, 1983).…”

cDNA cloning of myosin rod and the complete primary structure of myosin heavy chain of walleye pollack fast skeletal muscle

“…Lu and Lehrer reported that there are three highly conserved cysteine residues in rabbit skeletal S2 at position a at the 68th, 110th, and 412nd residues, which could also be found in pollack S2 at the same positions. 21 Systematic study with synthetic model peptides revealed that the stability of a double‐stranded coiled‐coil can increase significantly, when cysteine residues are placed in heptad positions d and form a disulfide bond. 20,21 In addition, when the disulfide bond is artificially introduced by 5,5’‐dithio‐bis(2‐nitrobenzoic acid) into the hydrophobic interface between two α‐helices, it considerably increases protein stability without a change in protein structure.…”

“… 21 Systematic study with synthetic model peptides revealed that the stability of a double‐stranded coiled‐coil can increase significantly, when cysteine residues are placed in heptad positions d and form a disulfide bond. 20,21 In addition, when the disulfide bond is artificially introduced by 5,5’‐dithio‐bis(2‐nitrobenzoic acid) into the hydrophobic interface between two α‐helices, it considerably increases protein stability without a change in protein structure. 22 However, functional significance of these conserved thiols in S1 heavy chain has remained unsolved.…”

cDNA cloning of myosin heavy chain from white croaker fast skeletal muscle and characterization of its complete primary structure

“…There are six cysteine residues in myosin rod, three highly conserved in myosin S2 and three in LMM 27 . It is known that there are three highly conserved cysteine residues at position a in myosin rod.…”