Student Research Project
Structure and function of heat shock proteins

Student
Denise Krawitz
Major: Molecular and Cellular Biology
Future Plans: Graduate school

Professor
Elizabeth Vierling, Associate Professor, Department of Biochemistry, University of Arizona, Tucson

When organisms are exposed to temperatures 5-10o C above normal growth conditions, a change in gene expression results in the synthesis of a set of proteins called heat shock proteins (HSPs). This phenomenon, referred to as the heat shock response, is highly conserved, occurring in all organisms studied, from bacteria to humans. A current hypothesis states that HSPs are critical to an organism's ability to survive heat stress. This is an important consideration for all organisms, especially plants, which do not have the ability to seek cooler surroundings. Exactly how HSPs function is not fully understood; however, several HSPs have recently been classified as molecular chaperones. Molecular chaperones are proteins that assist in the correct folding and assembly of newly synthesized or denatured proteins. Due to thermal denaturation and inactivation of proteins during heat shock, molecular chaperone activity may be critical in helping proteins reattain their active conformations at higher temperatures.

We are interested in understanding how a specific group of HSPs, the small HSPs (smHSPs), help protect organisms from heat shock. We have shown that PsHSP18.1, a smHSP from the plant Pisum sativum, can function as a molecular chaperone in vitro to protect enzymes from aggregation or to reverse enzyme inactivation at high temperature. PsHSP18.1 and other smHSPs form oligomeric (multi-united) protein complexes in vivo, and these higher molecular weight structures have been proposed as the functional form of the proteins. Because both oligomeric structure and function seem to be conserved in smHSPs, it is hypothesized that highly conserved amino acid residues in the protein play a critical role in smHSP structure and/or function.

To test this hypothesis, we have constructed site-directed mutants of the PsHSP gene using gene engineering techniques. We have changed either single amino acids in the protein or deleted a part of the protein. We can now examine the mutated proteins to determine if these changes removed or altered important parts of the protein necessary to retain its structure or activity. We have found that the mutant proteins created either fail to assemble into their oligomeric form and/or have altered molecular chaperone activity. This approach is frequently used by biochemists to investigate relationships between protein structure and function. Since we have mutants that remain monomeric, we are now in a position to test the hypothesis that the higher molecular weight complex is in fact the functional form of this smHSP. Continued analyses of these and other altered proteins may give us insight as to how smHSPs function, and will increase our understanding of how cells and organisms survive elevated temperatures.

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