Lawrence B. Smillie

Structures of the troponin C regulatory domains in the apo and calcium-saturated states.

Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction.

Structural interpretation of the two-site binding of troponin on the muscle thin filament.

Abstract Conditions are described for the quantitative removal of amino acid residues 274 to 284 from rabbit muscle α-tropomyosin with carboxypeptidase A. The product, non-polymerizable tropomyosin, has a much reduced affinity for the tropomyosinbinding fragment CB1 (residues 1 to 151) of troponin-T. Iodination of α-tropomyosin and non-polymerizable tropomyosin by 125 I and lactoperoxidase was carried out in the presence and absence of CB1. Following tryptic digestion and peptide mapping, the radioactivities of the labeled tyrosine peptides were compared. In the presence of CB1, tyrosine residues 261 and 267 were iodinated only to the extent of 30 to 40% as compared with the same tyrosine residues in the absence of CB1, All other tyrosine residues (60, 162, 214 and 221) were iodinated to a similar level in the absence or presence of CB1. With non-polymerizable tropomyosin, the presence of CB1 had a much reduced effect on the level of labeling of the tyrosine residues. We conclude that the highly helical region of troponin-T (residues 71 to 151) binds close to or at the COOH-terminal end of the tropomyosin molecule. Taken together with other considerations and recent observations, the results can be interpreted in terms of the two-site model for troponin attachment to the thin filament. A calcium-insensitive site would involve interaction of the highly helical CB2 region of troponin-T (residues 71 to 151) and the COOH-terminal region of tropomyosin (residues 258 to 284) and perhaps the NH 2 -terminal overlap region (residues 1 to 9). A calcium-sensitive site would involve the interaction of troponin-T in the neighborhood of cysteine 190 of tropomyosin in F-actin-tropomyosin assemblies both directly and indirectly through the association of its COOH and NH 2 -terminal regions with the troponin-I and C components.

Rabbit skeletal α-tropomyosin chains are in register

Abstract An intramolecular disulfide bridge can be formed between the two single cysteine residues of the coiled α-tropomyosin chains in both unmodified and cross-linked protein preparations. In neither case was there an increase in molecular weight as would be expected if the disulfide formation was intermolecular. Model building studies show clearly that this can only occur if the chains are in parallel and in register rather than in a staggered conformation. This conclusion indicates that the radius of the coiled-coil is variable and that the head-to-tail aggregation of tropomyosin molecules involves an overlap of both COOH-termini of one molecule and both NH 2 -termini of another.

Non-polymerizable tropomyosin: Preparation, some properties and F-actin binding

Summary Conditions are described for the nearly quantitative removal of residues 274–284 from the COOH-terminus of rabbit cardiac tropomyosin. The product is monomeric (Mr = 64,000) even at low ionic strength. The circular dichroism melting profile is only marginally different from the intact protein. The failure of the non-polymerizable tropomyosin to bind to F-actin, even at elevated MgCl2 concentrations, illustrates the importance of the head-to-tail interaction of tropomyosin molecules in their cooperative binding to the thin filament structure.

Amino acid sequences of rabbit skeletal β- and cardiac tropomyosins

Tropomyosin is an essential component of the actin-linked calcium regulatory systems in vertebrate skeletal and cardiac muscles. In skeletal muscle there are two major forms of the protein chain, designated (Y and 0, which can be separated by SDS-polyacrylamide gel electrophoresis [l-3] or by ion-exchange chromatography on CM-cellulose in urea buffers [3,4]. The ratio of the cyand @-tropomyosins appears to be related to muscle type with fast white muscles having a higher proportion of the (Y component (a

Amino acid sequence of Streptomyces griseus protease B, a major component of pronase

ratio of -3 or 4 : I). In slow red and smooth muscles this ratio is close to 1 [3,5,6]. Recent evidence indicates that ff-tropomyosin in skeletal muscles is restricted to type II cells [7]. Although it has not yet been established that P-tropomyosin is found only in type I cells, the results clearly imply that the varying ratios are a reflection of the relative proportions of the two classes of cells in various muscle types. In cardiac tissue, the 0 component appears to be absent in small and fast-beating hearts and present at low levels (1 S-20% of the total) in larger mammals [8 1. In addition rabbit cardiac tropomyos~ is similar to pure skeletal cr-tropomyosin in terms of its ammo acid composition and thiol content, behavior on SDS-polyacrylamide gels, immunological properties and peptide mapping [3,5,9]. The amino acid sequence analysis of rabbit skeletal cr-tropomyosin [lo-121 and its interpretation by several workers has permitted rational explanations for the stabilization of its coiled-coil structure [10,13-161, for its head-to-tail a~regation as long foments [15,17,18], for its interaction with 7 actin monomers along each of the two strands of F-actin

Amino acid sequence of pilin isolated from pseudomonas aeruginosa PAK.

Summary Streptomyces griseus Protease B is a close homologue of Protease A, another serine protease isolated from Pronase. Homology based on identity of residues in the two enzymes is 61%. Extensive identical sequences are found in the vicinities of histidine-57, aspartic acid-102, serine-195, the two disulfide bridges, the NH2-terminal and COOH-terminal ends as well as in the region of the presumed substrate binding sites. However, certain regions of the Protease B sequence are markedly different and include a heptapeptide insertion between residues 88 and 89 and a tetrapeptide deletion between residues 129 and 136 when compared with Protease A. These differences are probably responsible for the remarkable stability of Protease B in concentrated solutions of urea and guanidine hydrochloride.

Amino acid sequence of equine platelet tropomyosin: Correlation with interaction properties

Pseudomonas aeruginosa strain PAK have polar pili which are flexible filaments of about 5.4 nm diameter and 2.5 pm average length [l-3]. These pili consist of a single subunit, pilin, which was originally reported to have a A4, of 18 100 on the basis of SDS-polyacrylamide gel electrophoresis and amino acid compositional studies [4]. The sequence of the first 22 amino acids of the Nterminal region of PAK pilin have been reported [5] and shown to be strikingly homologous to the N-terminus of pili [6] isolated from Moraxella nonliquefaciens and Neisseria gonorrhoeae [7]. The Nterminal amino acid is the unusual N-methylphenylalanine [7,8]. [9]. This strain was kindly provided by Dr D.E. Bradley (Memorial University, St. John’s, Newfoundland).

Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

Equine platelet β tropomyosin (247 residues), like rabbit skeletal muscle α tropomyosin (284 residues) has a repeating pattern of amino acid residues characterisitic of a coiled‐coil structure. When compared with the muscle protein, it is extended by 5 residues at the NH2‐terminus and possesses two 21 residue deletions (positions 23–43 and 60–80 of the muscle sequence). The two proteins are highly conserved from residues 81–260, but are significantly different at their COOH‐termini (residues 261–284). These differences in platelet tropomyosin can be correlated with its diminshed head‐to‐tail polymerization, a weaker interaction with F‐actin and a reduced affinity for muscle troponin and the T1 fragment of troponin‐T.

Remarkable homology about the disulfide bridges of a trypsin-like enzyme from Streptomyces griseus.

In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be severely compromised by acid pH as in the ischemia of myocardial infarction and other cardiac myopathies. Recent reports (Westfall, M. V., and Metzger, J. M. (2001) News Physiol. Sci. 16, 278–281; Li, G., Martin, A. F., and Solaro, R. J. (2001) J. Mol. Cell. Cardiol. 33, 1309–1320) have indicated that the reduced Ca2+ sensitivity of cardiac contractility at low pH (≤pH 6.5) is attributable to structural difference(s) in the cardiac and skeletal inhibitory components (TnIs) of their troponins. Here, using a reconstituted Ca2+-regulated human cardiac troponin-tropomyosin actomyosin S1 ATPase assay, we report that a single TnI mutation, A162H, restores Ca2+ sensitivity at pH 6.5 to that at pH 7.0. Levels of inhibition (pCa 7.0), activation (pCa 4.0), and cooperativity of ATPase activity were minimally affected. Two other mutations (Q155R and E164V) also previously suggested by us (Pearlstone, J. R., Sykes, B. D., and Smillie, L. B. (1997) Biochemistry 36, 7601–7606) and involving charged residues showed no such effects. With fast skeletal muscle troponin, a single TnI H130A mutation reduced Ca2+sensitivity at pH 6.5 to levels approaching the cardiac system at pH 6.5. These observations provide structural insight into long-standing physiological and clinical phenomena and are of potential relevance to therapeutic treatments of heart disease by gene transfer, stem cell, and cell transplantation approaches.