The role of key amino acids in the cold-adaptation of the coiled-coil protein tropomyosin

The structural basis of cold-adaptation in a rod-shaped (α-helical, coiled-coil) protein tropomyosin was investigated by site-directed mutagenesis. An a -type tropomyosin from Atlantic salmon skeletal muscle having twenty amino acid substitutions compared to a warm-blooded counterpart (rabbit) was used as a model. Two adaptive-strategies were elucidated. The conformational stability of tropomyosin was shown to be enhanced by the presence of a polar amino acid, threonine-179, within the hydrophobic core and the presence of a pair of closely-spaced glycines at positions 24 and 27. The specific details are outlined below in point form: -- 1) Four mutants of Atlantic salmon fast skeletal muscle alpha-tropomyosin were engineered using the QuikChange Lightning site directed mutagenesis kit. Mutations were chosen in order to investigate the role of a pair of unique glycines near to the amino-terminal end as well as threonine 179 which occurs in a core position of the coiled coil. The four mutants were: Gly24Ala, Gly27Ala, Thr179Ala and a double mutant, Gly24Ala/Gly27Ala. Mutations were confirmed by DNA sequencing. -- 2) Recombinant (mutated and non-mutated) tropomyosins were obtained by expression in BL21 cells and induction with isopropylthiogalactopyranoside. Enriched protein was isolated, without exposure to organic solvents or high temperatures, by salt-induced precipitation and chromatography on ion exchange column and hydroxyapaptite columns. -- 3) Far-UV circular dichroism was used to investigate the conformational stability of the recombinant tropomyosins (0.1M NaCI, 20mM sodium phosphate , 1-2 mM dithiothreitol, 0.01 % mass/vol NaN₃). The observed melting temperatures of the three glycine mutants were similar to each other and that of the non-mutated recombinant tropomyosin: Gly24Ala, 36.9 °C; Gly27Ala, 37.3°C; Gly24AlaGly27Ala, 38.1°C and non-mutated, 37.0°C. However, the Thr179Ala mutant showed significant stabilization, 40.7°C. -- Melting profiles of the four tropomyosin mutants and non-mutated recombinant tropomyosin showed that the four mutants displayed more cooperative unfolding profiles compared to the non-mutated protein. -- 4) Limited chymotrypsin digestion (buffer: 50 mM NH₄HCO₃, 0.1 M NaCl, 1 mM DTT, pH 8.5), as monitored by SDA PAGE, revealed that the non-mutated tropomyosin is more susceptible to proteolysis than the Thr179Ala mutant (37°C, ~1:500 enzyme:substrate mole ratio). After 30 min, none of the intact non-mutated protein was detected in the reaction mixture whereas not all of the Thr179Ala mutant had been digested. At ~25 and ~10°C, the difference in the rate of digestion between the two, was not as significant. -- 5) From points 3 and 4 it can be concluded that threonine in the 179 position of salmon tropomyosin is destabilizing compared to the alanine in the same position of rabbit skeletal alpha-tropomyosin. -- 6) Sequencing of two fragments from the chymotrypsin digestion of non-mutant recombinant tropomyosin indicates that the initial cleavage site is between Leu 11 and Lys 12. -- 7) Omp-T digestion (buffer: 0.1 M NaCl, 50 mM sodium phosphate, 5 mM EDTA, 1 mM DTT, pH 7.0) patterns, as monitored by SDS PAGE, were compared between GLY24Ala, Gly27Ala, Gly24AlaGly27Ala, and non-mutated tropomyosin at ~10, ~25, or ~37°C. At all three temperatures, non-mutated tropomyosin was digested faster compared to the glycine mutants, which were different to each other. The observed rate of breakdown decreased in the order: Gly27Ala>Gly24Ala>Gly24AlaGly27Ala, indicating that both glycines influence the conformational stability of the amino-terminal region and that Gly24Ala is more influential than Gly27Ala. -- Note: Some of the above findings were reported in preliminary form at this year's Biophysical Society Meeting (Fudge, K.R. and Heeley, D.H. (2011) A mutant of Atlantic salmon fast muscle tropomyosin. 55th Biophysical Society (Baltimore)). A full manuscript is in preparation for submission to Biochemistry