Joyce R. Pearlstone

Amino acid sequence of pilin isolated from pseudomonas aeruginosa PAK.

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

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.

Periodicity of α-helical potential in tropomyosin sequence correlates with alternating actin binding sites

By averaging the α-helix parameters of Chou & Fasman (1974) over extended segments of the α-tropomyosin sequence, the maxima and minima of the 7-fold periodicity initially detected by Parry (1975) are observed to correspond approximately to the outer non-polar positive zones of the alternating β and α actin binding sites, respectively, described by McLachlan & Stewart (1976a). The periodicity is well developed in the NH2-terminal and central regions of the molecule but becomes progressively less distinctive towards the COOH-terminus. Initial cleavage points by trypsin and chymotrypsin occur close to minima in the averaged α-helical parameters.

Effects of high pressure and temperature on the wild-type and F29W mutant forms of the N-domain of avian troponin C

The N-domain of troponin C (residues 1-90) regulates muscle contraction through conformational changes induced by Ca2+ binding. A mutant form of the isolated domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressure and temperature changes. Here we set out to determine whether the point mutation itself has any effects on the protein structure and its stability to pressure and temperature in the absence of Ca2+. Molecular dynamics simulations of the wild-type and mutant protein structures suggested that both structures are identical except in the main chain and the loop I region near the mutation site. Also, the simulations proposed that an additional cavity had been created in the core of the mutant protein. To determine whether such a cavity would affect the behavior of the protein when subjected to high pressures and temperatures, we performed 1H-NMR experiments at 300, 400, and 500 MHz on the wild-type and F29W mutant forms of the chicken N-domain troponin C in the absence of Ca2+. We found that the mutant protein at 5 kbar pressures had a destabilized beta-sheet between the Ca2+-binding loops, an altered environment near Phe-26, and reduced local motions of Phe-26 and Phe-75 in the core of the protein, probably due to a higher compressibility of the mutant. Under the same pressure conditions, the wild-type domain exhibited little change. Furthermore, the hydrophobic core of the mutant protein denatured at temperatures above 47 degrees C, while the wild-type was resistant to denaturation up to 56 degrees C. This suggests that the partially exposed surface mutation (F29W) significantly destabilizes the N-domain of troponin C by altering the packing and dynamics of the hydrophobic core.

Structure and Stability of Wildtype and F29W Mutant Forms of the N-Domain of Avian Troponin C Subjected to High Pressures

The N-domain of troponin C (residues 1–90) regulates muscle contraction through conformational changes induced by Ca2+ binding. A mutant form of this domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressure and temperature changes. In this study we examined the effect of the point mutation on the protein structure and its stability to pressure. We performed 1-D and 2-D 1H-NMR experiments at 300, 400, and 500 MHz on the wildtype and F29W mutant forms of the N-domain of chicken troponin C in the absence of Ca2+. We found that the mutant protein at 5 kbar pressures had a destabilized βl-sheet between the Ca2+-binding loops, an altered environment near Phe 26, and reduced local motions of Phe 26 and Phe 75 in the core of the protein, probably due to a higher compressibility of the mutant. Under the same pressure conditions, the wildtype protein experienced little effect. These results suggest that the surface mutation (F29W) significantly destabilizes the N-domain of troponin C by altering the packing and dynamics of the hydrophobic core.