Conformational Dynamics of Mitochondrial Hsp70

Abstract

Recently, we have investigated the conformation of Mitochondrial Heat Shock Protein 70 (mtHsp70), a protein involved in the import of proteins into mitochondria and in protein folding in the mitochondrial matrix. To investigate the distribution of conformations in mtHsp70 molecules under different conditions, we performed single-pair Forster resonance energy transfer (spFRET) experiments using burst analysis and pulsed interleaved excitation (PIE). The FRET efficiency is a very sensitive measure for the distance between donor and acceptor on the scale of 2-10 nm and thus provides information over the conformation of mtHsp70 during its conformational cycle. To extract quantitative information about the homogeneity of the observed states, we used probability distribution analysis on the burst analysis data.MtHsp70 consists of two functional domains: the N-terminal nucleotide binding domain and the C-terminal substrate binding domain with a lid to hold the substrate peptide. Experiments were performed using FRET sensors for the interdomain distance and for the lid conformation with different nucleotides as well as in the absence or presence of peptide substrates and cochaperons. Our results show a surprisingly heterogenous conformational distribution for ADP bound mtHsp70, opposed by a more homogenous state with an open lid and close domain in the ATP bound state. Substrate binding in the presence of the cochaperone Mdj1 results in the undocking of the domains and closure of the lid holding the peptide. To investigate the conformational dynamics on a longer timescale (10 ms to 10 s) , we have performed spFRET TIRF experiments on immobilized molecules. We utilized two techniques to avoid immobilization artifacts. Either the protein was encapsulated in lipid vesicles or the substrate peptide was immobilized. The dynamics for the two sensors in the different states were compared.