Flexibility of the myosin heavy chain: direct evidence that the region containing SH/sub 1/ and SH/sub 2/ can move 10 /Angstrom/ under the influence of nucleotide binding

Journal Article · Tue Dec 13 00:00:00 EST 1988 · Biochemistry; (United States)

OSTI ID:6245223

Huston, E E; Grammer, J C; Yount, R G

Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF/sub 1/) could be oxidized to a disulfide bond by treatment with a 2-fold excess of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in the presence of MgADP. The resulting characteristic changes in the ATPase activities of SF/sub 1/ and the fact that MgADP was stably trapped at the active site, suggested that the two thiols cross-linked were SH/sub 1/ (Cys-707) and SH/sub 2/ (Cys-697) from the myosin heavy chain. To verify this suggestion, SF/sub 1/, after DTNB treatment as above, was treated with an excess of N-ethylmaleimide to block all accessible thiols. The single protein disulfide produced by DTNB oxidation was reduced with dithioerythritol and the modified SF/sub 1/ internally cross-linked with equimolar (/sup 14/C)p-phenylenedimaleimide (pPDM) in the presence of MgADP. After extensive trypsinization, the major /sup 14/C-labeled peptide was isolated, characterized, and shown to be Cys-Asn-Gly-Val-Leu-Gly-Ile-Arg-Ile-Cys-Arg, in which the two cysteines were cross-linked by pPDM. This peptide is known to contain SH/sub 2/ and SH/sub 1/ in this order and to come from residues 697-708 in the rabbit skeletal myosin heavy chain. Parallel experiments with (/sup 14/C)pPDM and unmodified SF/sub 1/ similar to those above gave an identical SH/sub 1/, SH/sub 2/ tryptic peptide, verifying earlier labeling results. These combined results demonstrate that SH/sub 1/ and SH/sub 2/ cross-linked by pPDM (12-13 /Angstrom/, S to S) or by oxidation with DTNB (2 /Angstrom/, S to S) can move a minimum of 10 /Angstrom/ under the influence of nucleotide binding. Because these residues are separated by only nine amino acids in the primary sequence, this small section of the heavy chain must possess extraordinary flexibility.

• Research Organization: Washington State Univ., Pullman (USA)
• OSTI ID: 6245223
• Journal Information: Biochemistry; (United States), Vol. 27:25
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
CROSS-LINKING
MYOSIN
NEM
BIOLOGICAL EFFECTS
AMINO ACID SEQUENCE
ATP-ASE
BENZOIC ACID
ENZYME ACTIVITY
IMIDES
OXIDATION
ACID ANHYDRASES
ANTIMITOTIC DRUGS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DRUGS
ENZYMES
GLOBULINS
HYDROLASES
LABELLED COMPOUNDS
MOLECULAR STRUCTURE
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHOHYDROLASES
POLYMERIZATION
PROTEINS
RADIOSENSITIZERS
550201* - Biochemistry- Tracer Techniques