Don't FRET the exchange rate: the study of subunit exchange between oligometric complexes of ZmHsp17.0-CII using fluorescence resonance energy transfer

Chris T. Nau, The College of Wooster

Abstract

Small heat shock proteins (sHsp) are a group of low weight (~30 kD) molecular chaperones belonging to the _-crystallin superfamily. While originally identified as being expressed during heat shock, additional studies have suggested that sHsps are expressed in response to a variety of cellular stresses and may be involved in more subtle roles in vivo. While found almost ubiquitously, plants express a disproportionately large number of sHsps that are classified into more than six distinct categories based on cellular localization. Important to this study are two cytosolic groups of sHsps; class I, that has been extensively studied, and class II, of which little is specifically known. Several mechanisms of chaperone activity have been proposed for sHsps, one crucial component of which appears to be the dissociation/reassembly of sHsp oligomers. This project sought to further understand this dynamic nature of oligomeric structures by determining the rate of subunit exchange of a class II sHsp, ZmHsp17.0, using fluorescence resonance energy transfer (FRET). FRET techniques used in this analysis differed from previously published work in that they measured the decrease in FRET from a sample that had been labeled with both dyes that was subsequently mixed with unlabeled sHsp, rather than monitoring the increase of FRET from the mixing of two alternatively labeled species. This method was found to be a more sensitive way to measure subunit exchange, leading to the determination of two rate constants for subunit exchange: k1 = 4.7 +/- 7.5 min-1 and k2 = 0.016 +/- 0.0013 min-1, thought to be representative of dimeric and monomeric exchange, respectively. The identification of two subunits involved in exchange has not been previously observed and may suggest a subtle role of the monomer in overall chaperone activity.