Selective cleavage at lysine of the 50 kDa‐20 kDa connector loop segment of skeletal myosin S‐1 by endoproteinase Arg‐C

Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultsmentioning

confidence: 96%

Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex

Kasprzak¹,

Chaussepied²,

Morales³

1989

“…Moreover, the hypersensitive tryptic cleavage site at or around Lys(253) [next to another lysine at residue 254 and near Lys(257)I is also attacked by this enzyme. Arg-C endoproteinasecatalyzed cleavage at lysine has been reported previously for a LysLys-Lys cluster within skeletal myosin S-1 (Bertrand et al, 1989).…”

Section: Biochemistrymentioning

confidence: 90%

Bacteriophage T4 gene 32 protein: Modulation of protein-nucleic acid and protein-protein association by structural domains

Casas‐Finet¹,

Karpel²

1993

The cooperative binding of bacteriophage T4 gene 32 protein to single-stranded nucleic acids is dependent on homotypic protein-protein interactions between the N-terminus of a protein monomer with the core domain of an adjacent protein. In a previous report [Casas-Finet et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1050-1054], we demonstrated that synthetic peptides corresponding to various portions of the N-terminal B-domain (residues 1-21) formed a 1:1 complex with core domain and identified a sequence, residues 3-5, Lys-Arg-Lys-Ser-Thr (the LAST motif) strongly homologous to a sequence within the central portion of protein (core domain) that was likely to function in nucleic acid binding. On the basis of these observations, we proposed a model where cooperative binding involves an exchange of intramolecular protein-protein interactions involving the internal LAST sequence for intermolecular protein-protein interactions utilizing the N-terminal LAST sequence. In this paper, we have tested various predictions of the model, and utilizing several proteases, further have defined the domain structure of 32 protein. The interaction of peptides containing LAST sequences with 32 protein qualitatively reduces its binding cooperativity, indicating that the peptides bind at the same site within the core domain as the N-terminus of an adjacent intact protein bound to the polynucleotide lattice. As expected, these peptides bind to nucleic acids. The N-terminus of 32 protein is predicted to be largely alpha-helical, and the circular dichroism spectrum of a peptide corresponding to residues 1-17 is consistent with this prediction. On the basis of the magnitude of protein tryptophan fluorescence quenching, the conformational change in 32 protein brought about by LAST peptides may be similar to that effected by oligonucleotides. As predicted by our model, in the presence of interacting peptide, the binding of 32 protein to oligonucleotide becomes salt-dependent. Arg-C endoproteolysis of intact 32 protein indicates that the loss of as few as three or four amino acids from the N-terminus appears to eliminate binding cooperativity, although the remainder of the N-terminal B-domain appears to protect the core from proteolysis. In contrast, this enzyme will catalyze the breakdown of trypsin-generated core domain, which lacks the first 21 residues of the protein. Thus, the presence of residues 4/5-21 attached to core alters its conformation and/or accessibility to protease. Poly(dT) inhibits this digestion, whereas the presence of N-terminal peptide accelerates proteolysis, in agreement with our model.(ABSTRACT TRUNCATED AT 400 WORDS)

“…In contrast to the COOH-terminal 22-kDa fragment issued from V8 protease proteolysis, the 21-kDa fragment was unable to cross-link to actin. These two fragments differ only in their NH2-terminal sequences; the former begins at Gly632, and the latter, at Lys642 (Chaussepied et al, 1983;Bertrand et al, 1989a). This suggests that cross-linking takes place on the 10-residue difference segment located at the NH2 terminus of the 22-kDa fragment.…”

Section: Effect Of Regulatory Proteins On Glutaraldehyde-inducedmentioning

confidence: 99%

Tropomyosin Inhibits the Glutaraldehyde-Induced Cross-Link between the Central 48-kDa Fragment of Myosin Head and Segment 48-67 in Actin Subdomain 2

Bonafé¹,

Mathieu²,

Kassab³

et al. 1994

The glutaraldehyde-induced cross-linking of the F-actin-myosin head (S1) complex, previously described [Bertrand et al. (1988) Biochemistry 27, 5728-5736], was investigated in the presence of tropomyosin (Tm) alone or associated with troponin (Tn), at a Tm-Tn/actin/S1 molar ratio of 1:7:3. Among the two acto-S1 cross-linked products with apparent masses of 165 and 200 kDa generated in the absence of the regulatory proteins, only the 165-kDa adduct was formed in the presence of Tm. An identical result was obtained with and without Tn regardless of the presence of Ca2+ and/or Mg(2+)-ADP. The abolition of the 200-kDa cross-linked acto-S1 species was independent of the S1/actin ratio since even a 3-fold excess of S1 over actin, sufficient for fully turning on the thin filament, could not restore the 200-kDa covalent complex. In addition, the acto-S1 contacts cross-linked in either the 165- or 200-kDa product were not involved in the Ca(2+)-linked regulation of the acto-S1 ATPase activity, as the enzymatic activities of both types of complexes were regulated to the same extent by Ca2+/EGTA, in the presence of the regulatory proteins. Cross-linking experiments performed with [14C]glutaraldehyde showed that both covalent complexes were composed of 1 mol of actin bound to 1 mol of S1 heavy chain. The use of proteolytic actin or S1 derivatives together with the direct proteolysis of the acto-S1 covalent adducts revealed that Tm abolished the cross-link between the central 48-kDa fragment of the S1 heavy chain and Lys50 of actin subdomain 2 that is responsible for the formation of the 200-kDa entity, while it did not affect the cross-link between the S1 heavy chain segment of residues 636-642 and Arg28 of actin that generates the 165-kDa derivative. These results provide experimental clues for the interaction of S1 with actin subdomain 2 and show that this contact is implicated in the weak acto-S1 binding state. Furthermore they demonstrate the ability of Tm to affect the structure of actin subdomain 2 even in the presence of S1 bound in the rigor state.