Consequences of placing an intramolecular crosslink in myosin S1

Abstract

This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, ''S1,'' of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4°C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this ''path of influence'' might be.S ome years ago, we reported on the possibility of emplacing a new intramolecular crosslink in myosin S1 (1, 2) by using a then newly invented reagent, dibromobimane (3). Extending this work has now led to results of interest beyond the original objective. Our early work, based on identifying N-terminal end groups, indicated that CNBr cutting generated an H-shaped fragment whose ''crossbar'' is a dibromobimane joining Cys-522 and Cys-707. In the present work, this identification is strengthened by amino acid sequencing from Phe-496 to Glu-509 (interrupted by Trp-510) and from Glu-687 to Leu-711, further establishing the identity of the two cited cysteines. It was noted by Burke (4) that, although he could confirm the 522-707 crosslink at room temperature, the link appeared impossible at 0°C. His observation stimulated us to study the temperature dependence of the crosslinking. As we report here, the reaction with Cys-707 is rapid and depends little on temperature, but the subsequent crosslinking with Cys-522, achievable within 30 min at 25°C, requires a week at 0°C. Crystallography (5, 6) corresponding to near 0°C (as do all current images of S1) indicates an observable interthiol separation of about 23.49 A, compared with the 8-Å length of bimane (the next Cys in the sequence is even farther, at 41.85 Å). This suggests that, after anchoring at Cys-707, the yet unreacted function of dibromobimane and Cys-522 may engage in a slow temperature-dependent search for one another or else are brought together by a structural transition not evident in 4°C images. Although measurements of the ADP-S1 affinity with native S1 conform with the classic LoweyLuck (7) measurements on intact myosin, it is clear that the affinities are about one-third less with crosslinked S1, or with S1 reacted with monobromobimane (which reacts only Cys-707). Additionally, attaching either bimane alters the nuclear magnetic resonance arising in the P-atoms of the bound ␤-phosphate. These somewhat disparate o...

On the tryptophan residue of smooth muscle myosin that responds to binding of nucleotide

“…Thus we support early suggestions by Johnson et al (9), and by Hiratsuka (10) and the more definitive recent work, namely, the mutational work of Batra and Manstein (12), using the homology of Dictyostelium, and that of Yengo et al ‡ ‡ , applying a ''one Trp only'' strategy to smooth muscle myosin, and the recent spectral analysis of Park and Burghardt (14). Besides confirming recent work, application of a one substitution at a time strategy leads us to the finding, namely, that the enzymatic properties of W512F HMM are strikingly like those of smooth muscle myosin (26) whose ''SH1s'' (Cys-717s) have been reacted with thiol reagent.…”

“…In the reverse direction, reacting Cys-717 with a thiol reagent facilitates the ''leaving'' of bound MgADP (11,26). Also in the same direction, an NMR signal (the resonance of the 31 P of the ␤ phosphate of bound MgADP) is changed on reacting Cys-717 with a thiol reagent (14). In this perspective, in the presence of MgADP, we begin investigating an in-bound path of influence between position 512, position 717, and the active site.…”

Quantitative evaluation of the lengths of homobifunctional protein cross‐linking reagents used as molecular rulers

“…The final group ( Fig. 9; Table 5) consists of three short and nearly rigid cross-linking reagents: 1,5-difluro-2,4-dinitrobenzene (DFDNB; e.g., see Mayeux et al 1991;Herzig et al 1995;Krupenko et al 1995;Shoshan-Barmatz et al 1995;Herzig et al 1996), 4,4Ј-difluoro3,3Ј-dinitrodiphenylsulfone (DFDNPS; e.g., see Givol 1969;Modesto and Pesce 1971;Hsia et al 1984), and dibromobimane (bBBr; e.g., see Kim and Raines 1995;Bhattacharjee and Rosen 1996;Wu et al 1996;Clarke 1997, 1999;Konno et al 2000). The first two are amine reactive through nucleophilic aromatic substitution to give aryl amines.…”

Effects of Oxidative Stress on Behavior, Physiology, and the Redox Thiol Proteome ofCaenorhabditis elegans

“…The fact that we independently identified the same cysteine to be redox sensitive in two different myosin-isoforms (UNC-54, MYO-2) illustrates the general oxidation sensitivity of this specific cysteine residue. This is particularly significant because this cysteine is highly conserved from yeast to human myosin and is located adjacent to a second conserved cysteine, which has been proposed to be redox sensitive and involved in the ATPase activity of myosin (25). Oxidative inactivation of UNC-54, the major C. elegans myosin heavy chain required for locomotion and egg-laying, and MYO-2, which is exclusively expressed in the pharynx, might contribute to the observed peroxide-mediated defects in motility, pharyngeal pumping, and egg-laying.…”