Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.

Abstract

Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols (benzophenone-4- Communicated by Russell F. Doolittle, May 23, 1986 ABSTRACT The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (Si), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) Is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared In the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis 'was carried out in the presence of Mg2+ and ATP or Mg2e and adenosine 5-[,B, imidoltriphosphate (AdoPP[NHJP), a 70-kDa band, attribu-

Pathway for the communication between the ATPase and actin sites in myosin

“…Some analogs, such as 3'-O-3(-N-(4-azido,2-nitrophenyl)amino)propionyladenosine-5'-triphosphate (Szilagyi et al, 1979) or N-(4-azido,2-nitroanilino)ethyl diphosphate (Okamoto & Yount, 1983) wer e incorporated into the N-terminal fragment, while others, such as 3'-O-(4-benzoyl)-benzoic-adenosine-5'-triphosphate (Mahmood & Yount, 1984;Srivastava et al, 1986), N-(4-azido,2-nitroanilino)ethyl triphosphate (Nakamaye et al, 1985) or 1-fluoro-2,4-dinitrobenzene (Hiratsuka, 1986a) specifically labelled the central fragment. These results suggest that although the 27K fragment contains information for nucleotide adenine binding, part of the 50K fragment may be located at or near the Muno et al, 1987Lu et al, 1986Chaussepied et al, 1986f Chaussepied et al, 1988Hiratsuka, 1984Rajasekharan et al, 1987 active site. Both the heavy chain and the light chain may contribute to the ATP binding site, as recently r• in srnooth muscle myosin .…”

Domains, motions and regulation in the myosin head

“…In myosin, as in adenylate kinase, this loop motion could alternately block and open the entrance to the pocket and/or alter the affinity of the site for ATP and ADP (Fry et al, 1986), as well as serving the critical function of communicating nucleotide binding to the actin-binding site. Motion in the head due to nucleotide binding is revealed by effects on crosslinking: the reactive sulphydryl SH1 in the 20 k region can be linked to the 25 k region in the absence of ATP, and also to the 50k region in the presence of nucleotide (Lu et al, 1986). Trapping the nudeotide favours 20-50 k crosslinking (Rajasekharan et al, 1987), probably by immobilizing the.…”

Three-dimensional structure of myosin subfragment-1 from electron microscopy of sectioned crystals.

“…Biochemical and biophysical evidence for intramolecular motion in myosin is available . For example, nucleotide binding to the myosin head causes internal motion that is reflected by changes in the pattern of cross-links that can be formed with SH, and by changes in proteolytic susceptibility of bonds (Lu et al, 1986;Applegate and Reisler, 1984) . Weak and strong binding states of actin also suggest nucleotide induced motion that dramatically alters actin binding affinity (Eisenberg and Greene, 1980) .…”