The Conformational Dynamics of the Mitochondrial Hsp70 Chaperone

Abstract

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

“…Double-labeled DnaK(318,425) was expressed, purified and labeled as described in ref. [8]. This FRET sensor was designed to monitor the distance between the substrate binding domain and the nucleotide binding domain of DnaK, the major bacterial Hsp70.…”

“…Recently, we investigated the conformational cycle of the yeast mitochondrial Hsp70 Ssc1 using spFRET sensors and compared it to the prototype Hsp70 DnaK. [8] A FRET sensor was designed to monitor the distance between the NBD and SBD by introducing cysteine residues into DnaK at positions 318 and 425 and labeling them with Atto532 and Atto647N. The endogenous cysteine at position 15 of DnaK was mutated to alanine.…”

“…This residual width is attributed to slow dynamics of the linkers used to attached the fluorophores to the protein or to acceptor photophysics. [31,63] Thus, the width of the ATP state indicates that the protein conformation with ATP bound is rigid and very homo- [8] The experiments presented above were performed under static conditions. The ability of MFD to separate out subpopulations in single molecule experiments makes it possible to investigate DnaK under conditions where chaperoning cycling should occur.…”

“…Hence, the significant differences of the ADP state of Hsp70s from different organisms or organelles may be one indication of the evolutionary specialized functions of Hsp70s, which have adapted to their different cellular environments. [8] …”

“…Ha and coworkers showed that it is possible to perform FRET investigations with single-molecule sensitivity, [4] which has led to the wide spread use of single-pair FRET (spFRET) to investigate intra-and intermolecular distances and thereby conformations and dynamics in order to provide new insights into a number of biological questions. [5][6][7][8][9][10][11][12][13][14][15][16][17] SpFRET measurements can be performed either on immobilized molecules or on molecules in solution using a burst analysis. [18] In the solution-based experiments, the fluorescence of a dilute (of the order of 20-50 pm) solution of labeled molecules coming from the~fL volume of the focus of a confocal microscope is measured using a high numerical aperture objective.…”