Dr Craig Marshall

Biochemistry Department University of Otago P.O. Box 56 710 Cumberland St Dunedin 9054 , New Zealand Tel.: +64 3 479-7570 FAX: +64 3 479-7866 e-mail: craig.marshall@stonebow.otago.ac.nz

The Structural Basis of Cold-Adaptation in Proteins

Overall Aim of our Research

Our major goal in this work is to improve our understanding of the structural and functional adaptations of the enzymes that fit them for function in Antarctic fish and how these adaptations compare with those of related fish from more temperate waters. In the long-term we plan to examine a number of proteins so that we can produce a general picture of how low temperature affects protein structure and function. This work will also deepen our understanding of the mechanisms by which ligands interact with enzymes and will improve our knowledge of how proteins fold.

In addition to the general question of how proteins fold to allow activity at low temperatures, we are interested in the specific question of the nature of the structure of antifreeze glycopeptides. We wish to answer this question by growing crystals of these proteins and solving the structure by X-ray crystallography.

Proposed Research

Cold-adapted Proteins:

Proteins in Antarctic fish continue functioning despite being exposed to temperatures below that at which most proteins lose activity. Decreased temperatures have effects in two main areas in which we are interested; changes to ligand-binding, and the effects of temperature on overall protein stability.

Ligand-binding:

Much of the reduction of enzyme activity with temperature can be ascribed to a reduction of thermal motion. At low temperatures, binding of ligands is likely to be of a higher affinity, and the enzyme itself is less likely to "breathe". The consequence of increased affinity of ligand-binding is complex. Although the substrate may well bind more readily to an enzyme (possibly associated with a reduced K_m), the tightness of binding may inhibit the conformational changes necessary for the catalytic cycle to occur. Furthermore, once product is formed, it may have a higher affinity for the enzyme at lower temperatures, and thus be less likely to leave the complex, reducing the catalytic efficiency of the enzyme. We might expect then that enzymes that are adapted for activity in the cold will show reduced ligand-binding interactions; particularly binding to product. It is difficult to assess the nature of the binding interactions that are likely to be altered. Whether there is a selective loss of hydrophobic interactions, which tend to be favoured at low temperatures, hydrogen bond or van der Waals interactions, or instead a generalized reduction of binding interactions is something that can only be determined by experiment.

Protein Structure and Stability:

The general decrease in thermal motion at lower temperatures will also have important consequences for enzyme action. Catalytic activity in enzymes is almost always associated with movement of one sort or another. Indeed, the introduction of disulfide bridges into enzymes in an effort to increase their thermal stability has sometimes been associated with a loss of catalytic activity as the introduced bridges have been too effective in constraining thermal motion. Experience with proteins from thermophilic organisms where the requirement for increased thermal stability is paramount, has suggested that more efficient hydrophobic packing is an effective way to increase the thermal stability. These studies have helped to highlight the importance of hydrophobic interactions in determining protein folding.

Cold Adaptation:

Cold-adapted proteins are faced with the converse problem. The reduced temperature is likely to make them too stable and unable to undergo the conformational changes associated with catalysis. Determining the structure of cold-adapted LDH will allow us to determine the changes that are likely to be responsible for changing the thermal stability to allow conformational change; whether it be decreased efficiency of hydrophobic packing in the core of the protein, a reduction of hydrogen bonding or the introduction of electrostatic interactions serving to reduce the protein stability.

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The Sub-dermal Matrix Proteins from the Snail Fish

The snail fishes (genus Paraliparis) are characterized by a layer of jelly between the muscle and the skin so much so that they look like a fish in a condom. Snail fishes are typically found in deepwater and are widespread in deeper waters around the world. In Antarctic water a number of snail fish species are found, and it seems likely that many more species are likely to be discovered.

Preliminary work has characterized some of the proteins in this matrix (1, 2). In this work we propose to isolate and identify a number of the proteins from the subdermal matrix. Purified proteins will be partially sequenced and PCR primers constructed to allow the amplification of the corresponding cDNA for sequencing. The physiological role of these proteins will be investigated, particularly any ice-binding and thermal hysteresis activity.

1. Eastman, J.T., Hikida, R.S. & DeVries, A.L. (1994). "Buoyancy studies and microscopy of skin and subdermal extracellular matrix of the Antarctic snailfish, Paraliparis devriesi ."Journal of Morphology 220:85-101.

2. Jung, A., Johnson, P., Eastman, J.T. & DeVries, A.L. (1995). "Protein content and freezing avoidance properties of the subdermal extracellular matrix and serum of the Antarctic snailfish, Paraliparis devriesi ." Fish Physiology & Biochemistry 14:71-80.

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Group Members

Craig Marshall: Completed a BSc (Hons) and PhD in the Department of Biochemistry before gaining postdoctoral experience in Cambridge on an MRC-Wellcome Overseas Research Fellowship. After returning to the Department in 1991 as a Postdoctoral Fellow he was appointed as Lecturer in 1994. craig.marshall@stonebow.otago.ac.nz

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Grants

• 1997, Otago Research Grant, Ice-binding proteins in Antarctic fish.
• 1997, PGSF Antarctic Funding, Biochemical cold-adaptations of Antarctic fish.
• 1996, Lottery Health, Chromatographic equipment for protein fractionation.
• 1996, Lottery Health, Structural studies of bacterial release factors.
• 1995, Ross Dependency Research Committee, Structural studies of cold-adapted protein from Antarctic fish. Approval of project and logistical support to work in the Antarctic at Scott Base and McMurdo Base.
• 1995, Marsden Fund, Structural studies of proteins from cold-adapted Antarctic marine organisms.
• 1995, Otago Research Grant, Structural and kinetic studies of cold-adapted proteins from Antarctic marine organisms.
• 1995, Health Research Council Travel Grant.
• 1995, Otago Medical School Travel Grant.
• 1994, Lottery Health, Structural investigations of proteins.
• 1994 Otago Research Grant, Structural and kinetic studies of cold-adapted proteins from Antarctic marine organisms.
• 1994, Lotteries Health, Investigations of polypeptide chain release factors.
• 1993, Health Research Council, A structural investigation of the polypeptide chain release factors.

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