A FRET reporter of conformation in Hsc70

Abstract

Hsp70s are the most well conserved protein family known, and yet little of their activity within the cell is understood. Hsc70 is a constitutively expressed member of the Hsp70 family found in the cytosol of almost all eukaryotic cells. Hsc70 has many functions within the cell, including assisting with the folding of nascent peptides, targeting proteins for degradation, and remodelling protein complexes. All Hsp70s consist of two highly conserved domains, the nucleotide binding domain and the substrate binding domain, joined by a flexible linker region. The juxtaposition of the two domains is dependent on the nucleotide bound by the nucleotide binding domain. These conformations have been studied in vitro, but not in vivo, so it is not known exactly how conformational change contributes to functions within the cell. Many previous studies of Hsp70s have relied on the bacterial form, DnaK. In this study, Hsc70 constructs were produced based on previously made DnaK versions. Using these constructs, the nucleotide dependent conformational shifts of Hsp70s were probed by Förster Resonance Energy Transfer (FRET). To do this, fluorescent dyes were attached to each domain of the protein. This allowed direct comparisons to be made between Hsc70 and DnaK. In this study we showed that tris(2-carboxyethyl)phosphine behaved anomalously on gel filtration, compromising the labelling protocol. This was improved by a change of reductant and gel filtration conditions. Of the four sites tested for labelling, one potential site, Q520, was found to be totally refactory to labelling. One of the aims of this study was to find an Hsc70 construct that would produce a strong FRET signal, which could then be used in future single molecule in vivo studies. The variant E318C/T427C/C574S/C603S gave the strongest signal of the four variants tested. Analysis of this variant also showed that Hsc70 and DnaK conformations are not the same, with ADP bound DnaK spending less time with the linker region exposed to solvent than ADP bound Hsc70 does.