Thiol-specific cross-linkers of variable length reveal a similar separation of SH1 and SH2 in myosin subfragment 1 in the presence and absence of MgADP.
A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys(707)) and SH2 (Cys(697)) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH(2))(n)()SSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 A. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k(1)) and for cross-linking (k(2)) were measured by the decrease of the K(+)(EDTA)-ATPase activity and the decrease of the Ca(2+)-ATPase activity, respectively, and corrected for the different reactivities of C(n). Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellman's reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as >/=15 A, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k(2) for C(3), C(6), and C(7) in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628].
Kliche
W
W
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany. wkliche@zellbio.mpg.de
Pfannstiel
J
J
Tiepold
M
M
Stoeva
S
S
Faulstich
H
H
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D007700:Kinetics; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D011817:Rabbits; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
D000199:Actins; D003432:Cross-Linking Reagents; D004220:Disulfides; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D013438:Sulfhydryl Compounds; D000244:Adenosine Diphosphate; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D003545:Cysteine