Date of Award

8-2005

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Bruce D. Sidell

Second Committee Member

Paul D. Rawson

Third Committee Member

Gregory D. Mayer

Abstract

Parvalbumins are proteins that buffer intracellular calcium concentrations and facilitate muscle relaxation. They are important in fish white muscle, which powers burst-speed swimming. Expression of parvalbumin in white muscle of Antarctic fishes is higher than has been observed in temperate water fishes. At respective physiological temperatures, parvalbumins from temperate water and Antarctic fishes exhibit similar calcium dissociation constants. However, calcium dissociation constants for temperate water fish parvalbumins are lower than those from Antarctic fish species across a range of temperatures. This observation suggests that Antarctic fish parvalbumins differ structurally from those of temperate water fish species. This study addressed the hypothesis that Antarctic fish parvalbumins have been selectively modified to conserve function at cold body temperatures. Parvalbumin cDNA was isolated from white muscle of Antarctic icefish Chaenocephalus aceratus, and three distinct parvalbumin mRNA transcripts were identified. One rnRNA transcript is nearly identical to that predicted from amino acid sequence of native parvalbumin from C. aceratus, which was generated by mass spectrometry protein sequencing. This result indicates that C. aceratus white muscle predominantly expresses one parvalbumin rnRNA. Nucleotide sequence of the parvalbumin gene coding region was obtained via RACE, and protein primary structure was deduced. Analysis of inferred amino acid sequence indicates that parvalbumin from C. aceratus white muscle contains 109 amino acids, with a molecular size of 11.5 kDa, similar to parvalbumins from other fishes. Amino acid composition is also similar to other fish parvalbumins. Analysis of parvalbumin amino acid sequence suggests specific modifications in protein primary structure may permit conservation of parvalbumin function at cold temperatures. Comparison of parvalbumin sequence between C. aceratus and other fish species reveals that E-F hand cation-binding domains are highly conserved across species. Amino acid substitutions in parvalbumin from icefish appear to occur in regions of the molecule responsible for positioning mobile elements of protein secondary structure. Comparison of parvalbumin sequences from C. aceratus and Cyprinus carpio (common carp) reveals several amino acid differences in hinge regions of the protein that result in a decrease in amino acid side-chain bulk in parvalbumin from C. aceratus. Within the second hinge region (amino acids 35-40), protein from C. aceratus contains a serine residue at position 37 and an alanine residue at position 38; sequence from carp contains threonine at position 37 and serine at position 38. Within the third hinge region (amino acids 68-79), protein from C. aceratus contains a serine residue at position 72 and a glycine residue at position 74; sequence from carp contains a lysine residue at position 72 and an aspartate residue at position 74. These reductions in bulk of side-chains likely confer increased flexibility in the cold-adapted protein. The parvalbumin cDNA from C. aceratus white muscle was transfected into a bacterial expression system and expressed protein was purified to homogeneity. Calcium dissociation constants from expressed protein are indistinguishable from those of native parvalbumin when measured across a range of temperatures. Future investigations using site-directed mutagenesis will determine the precise amino acid modifications that permit parvalburnin function at cold temperatures.

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