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

Fudge, Korrina R. (2011) The role of key amino acids in the cold-adaptation of the coiled-coil protein tropomyosin. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (26MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

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.

Actions (login required)

View Item