Date of Award

12-2003

Level of Access

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Cashon

Second Committee Member

Michael E. Vayda

Third Committee Member

Dorothy E. Croall

Abstract

Teleost myoglobin (Mb) proteins from four fish species inhabiting different temperature environments were used to investigate the relationship between protein function and thermal stability. Mb was isolated from yellowfin tuna (homeothermal warm), mackerel (eurythermal warm), and the Antarctic teleost Notothenia coriiceps (stenothermal cold). Zebrafish (stenothermal tropical) myoglobin was expressed from cloned cDNA. N. coriiceps Mb cDNA has also been cloned, expressed at 20°C , and isolated from E. coli, but was not used in any of the functional and kinetic studies. These proteins differed in oxygen affinity, as measured by O2 dissociation rates and P50 values, and thermal stability as measured by autooxidation rates. Mackerel Mb had the highest P50 value at 20°C (3.7mm Hg), corresponding to the lowest O2 affinity, followed by zebrafish ( 1.Omm Hg), yellowfin tuna (1.Omm Hg), and N. coriiceps (0.6mm Hg). Oxygen dissociation rates and Arrhenius plots were similar between all teleost species in this study, with the exception of mackerel myoglobin, which was two fold faster at all temperatures tested. Myoglobin from the Antarctic teleost had the highest autooxidation rate (0.44 h-1), followed by mackerel (0.26 h-1), zebrafish (0.22 h-1), and yellowfin tuna (0.088 h-1). Primary structural analysis revealed residue differences distributed throughout the polypeptide sequences, making it difficult to identify, which, if any, residues contribute to structural flexibility. However, analysis of molecular dynamics trajectories indicates that Mb from the eurythermal mackerel is the most flexible protein within the D loop and FG turn, which correlates with the O2 affinity and kinetic data.

Included in

Biochemistry Commons

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