Digestion of tropomyosin with trypsin

Gorecka, Alicja; Drabikowski, W.

doi:10.1016/0014-5793(77)80072-0

Cited by 13 publications

(6 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fragments have also lost the ability to bind to actin, in agreement with the results of Gorecka and Drabikowski (1977) for fragments prepared by tryptic digestion. The N-terminal fragment does not combine with troponin but there is some evidence for an interaction between the C-terminal fragment and troponin.…”

Section: Discussionsupporting

confidence: 85%

Stability of Segments of Rabbit a-Tropomyosin

Woods¹

1977

Aust. Jnl. Of Bio. Sci.

View full text Add to dashboard Cite

Rabbit IX-tropomyosin was cleaved into two pieces at the cysteine residue of each chain. The products were separated by chromatography and characterized by amino acid analysis, molecular weight determination in benign and denaturing solvents, optical rotation and circular dichroism. When the cleavage reaction was carried out under mild conditions which preserve the two-chain structure there was considerable loss of IX-helix in each segment.Thermal stability studies, monitored by optical rotation and circular dichroism, showed that the transition temperature of the N-terminal fragment at pH 7·6 was approximately 17°C higher than that of the C-terminal fragment. In acid solutions there is little difference in the thermal stability of the two segments. The least stable part of the molecule is conchided to be between residues 133 and 205 and this includes the troponin-binding site. The relative stabilities found for segments of rabbit IX-tropomyosin differ from recent published conclusions and this may be a result of the different methods used to study the loss of the IX-helical conformation.The two tropomyosin fragments, unlike the parent tropomyosin, do not inhibit actomyosin adenosinetriphosphatase when mixed with troponin. The fragments did not show any of the aggregation properties of tropomyosin and did not combine with actin. The N-terminal fragment did not complex with troponin but there was some evidence for an interaction between the C-terminal fragment and troponin.

show abstract

Section: Discussionsupporting

confidence: 85%

Stability of Segments of Rabbit a-Tropomyosin

Woods¹

1977

Aust. Jnl. Of Bio. Sci.

View full text Add to dashboard Cite

show abstract

“…This increased unfolding in disulfide-cross-linked tropomyosin appears to be related to an increase in tryptic cleavage at certain sites (Gorecka & Drabikowski, 1977;Ueno, 1984).…”

mentioning

confidence: 96%

Effects of interchain disulfide crosslinks on the trypsin cleavage pattern and conformation of myosin subfragment 2

Lu¹,

Lehrer²

1984

Biochemistry

View full text Add to dashboard Cite

The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

“…However, the stability is not uniform along the length of the molecule as heat and salt denaturation studies, calorimetric measurements of the intact molecule as well as peptides, motions of tropomyosin in the crystal lattice, and preferential sites of proteolytic digestion show (Ooi, 1967;Wood, 1969;Pont & Woods, 1971;Eckard & Cowgill, 1976; 0006-296018 5 10424-3 305$0 1.50/0 Gorecka & Drabikowski, 1977;Pato & Smillie, 1978;Ueno & Ooi, 1978;Phillips et al, 1979Phillips et al, , 1981Edwards & Sykes, 1980;Pato et al, 1981;Williams & Swenson, 1981;Potekhin & Privalov, 1982;Stewart & Roberts, 1983;Ueno, 1984). The results of many experiments indicate that the COOH-terminal half of the molecule is less stable than the NH2-terminal half.…”

mentioning

confidence: 97%

“…A region of preferential instability surrounds Cys-190, interpreted to be due to localized chain separation (Betcher-Lange & Lehrer, 1978; Lehrer, 1978; Betteridge & Lehrer, 1983). The state of oxidation or type of modification of the cysteines can have long-range effects: it can alter the susceptibility to proteolysis in the region 60 residues toward the NH2-terminus (Gorecka & Drabikowski, 1977;Ueno, 1984); the NMR resonance of histidine-153, 5.5 nm toward the NH2-terminus, is changed (Edwards & Sykes, 1980), and the degree of polymerization that involves the extreme ends of the molecule is affected (Graceffa & Lehrer, 1980).…”

mentioning

confidence: 99%

Tropomyosin lysine reactivities and relationship to coiled-coil structure

Hitchcock‐DeGregori¹,

Lewis²,

Chou³

1985

Biochemistry

View full text Add to dashboard Cite

We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions.

show abstract

Digestion of tropomyosin with trypsin

Cited by 13 publications

References 13 publications

Stability of Segments of Rabbit a-Tropomyosin

Stability of Segments of Rabbit a-Tropomyosin

Effects of interchain disulfide crosslinks on the trypsin cleavage pattern and conformation of myosin subfragment 2

Tropomyosin lysine reactivities and relationship to coiled-coil structure

Contact Info

Product

Resources

About