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. 2000 Feb 15;97(4):1461-6.
doi: 10.1073/pnas.030523997.

Consequences of placing an intramolecular crosslink in myosin S1

K Konno  1 , K Ue, M Khoroshev, H Martinez, B Ray, M F Morales
Affiliations

Consequences of placing an intramolecular crosslink in myosin S1

K Konno et al. Proc Natl Acad Sci U S A. .

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 degrees 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.

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Figures

Figure 1
Figure 1
Distances between Cys-707 and other thiols in the 50 kDa domain (cyan) and the 20 kDa domain (red). Residue numeration applies to rabbit muscle (based on ref. and PDP code 2 mys).
Figure 2
Figure 2
Local conformation (expressed as CA-CA distances) before (red) and after (magenta) a dibromobimane crosslink (in CPK style) is emplaced. This image was constructed from ref. , by eliminating the ADP.BeFx ligand of Dictyostellium and replacing Thr-513 and Thr-688 with their homologs in rabbit, viz. Cys-522 and Cys-707.
Figure 3
Figure 3
Environment of bound ADP and emplaced dibromobimane. Except for select, otherwise specified, portions, the “trace” is rendered as an orange “oval,” and the β-strands as yellow arrows. The helix that connects Cys-707 and Cys-697 is in light magenta; it connects via a helix and loop (both cyan) to the third β-strand of the β-sheet. The “P-loop” (blue) of the ADP binding site connects to the fourth strand of this β-sheet. The emplaced dibromobimane, shown as attached only to Cys-707, interacts with a helix and loop (both magenta) by steric and hydrophobic contacts. This picture was created from PDB code, 1 mmd, as MMD.ADP.BeFx. ADP placement was obtained by superimposition of the trace of MMD.ADP.BeFx on the trace of MMD.DBB. Sequence numbers are those of rabbit myosin.
Figure 4
Figure 4
(A) Fluorescence of S1 (in arbitrary units) acquired by reaction with dibromobimane, depicted as a function of time. The different symbols represent measurements made at various temperatures: open circles, 0°C; open squares, 5°C; open triangles, 10°C; closed circles, 20°C; closed squares, 25°C; closed triangles, 30°C. (B) Percent of dibromobimane-labeled S1 crosslinked (measured by the amount of 105-kDa band that forms on union of the 20-kDa and 50-kDa domains), depicted as a function of time, to show the first-order nature of the reaction. Symbols are as in A.
Figure 5
Figure 5
Logarithm of the as-yet-uncrosslinked fraction of S1, u, as a function of time. Experimental measurements indicated that the maximum fraction of S1 that could be crosslinked was approximately 0.6. Symbols are as in Fig. 4A; rates are estimated from Fig. 4B.
Figure 6
Figure 6
Rate of crosslinking S1 as a function of reciprocal absolute temperature. These data yield a heat of activation of ca. 8.5 kcal.
Figure 7
Figure 7
The 31P chemical shift spectra of MgADP interacting with myosin S1 (in various states of derivatization) in the presence of a reference substance and an adenylate kinase inhibitor at 0°C, pH 7; N, native; M, derivatized with monobromobimane; D, derivatized with dibromobimane. (Inset) The −2-ppm peaks have been isolated and enlarged to show changes resulting from derivatization. Note that N shows no doublet structure but clearly generates a peak (see refs. –13).
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