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

• Main Author: Fudge, Korrina R.
• Format: Thesis
• Language: English
• Published: Memorial University of Newfoundland 2011
• Subjects: Atlantic salmon
• Online Access: https://research.library.mun.ca/6111/; https://research.library.mun.ca/6111/1/Fudge_KorrinaR.pdf; https://research.library.mun.ca/6111/3/Fudge_KorrinaR.pdf

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 ...