Functional characterization of natural occurring variants of the 70‐kDa heat‐shock protein, HSPA1A (567.10)

Seventy‐kDa‐heat‐shock proteins (Hsp70s) are molecular chaperones essential for protein homeostasis and cell survival under both physiological and pathological conditions. Given this vital role of Hsp70s, it is important to investigate whether naturally occurring variants of these proteins have altered functional properties. In this study, the functional outcome of four non‐synonymous single nucleotide polymorphisms present in different human populations on HSPA1A was tested. Site directed mutagenesis was used to generate the HSPA1A mutations into both both bacterial and mammalian expression vectors. Through the use of a malachite green colorimetric ATPase assay, the mutant proteins’ ability to hydrolyze ATP at the protein’s ATP‐binding domain was tested. All four proteins were found to be functionally differentiated from each other and the WT. Based on the Km values obtained, the WT had the highest affinity for binding to ATP, but based on the Vmax values, all the mutants were able to hydrolyze ATP at a much faster rate, with the S16P mutant showing nearly a 4‐fold increase from the WT. The mutant proteins’ ability to properly localize intracellularly within HEK‐293 cells was also tested by tagging HSPA1A to GFP, using fluorescent dyes to stain the mitochondria, lysosomes, and plasma membrane, and viewing where the proteins colocalized via confocal microscopy. Under normal growth conditions all four mutants were found to localize to these regions in a similar manner to the WT indicating that these mutations did not affect the protein’s ability to localize within the cell. Grant Funding Source : This project was supported by funds from CSUPERB and CSUF to NN, and HHMI to MS and NN