TIRF detection of conformational change in DnaK by single-molecule FRET

Abstract

Experiments pursuing the dynamics of single protein molecules present unique insights into structural mechanics and behaviour at the molecular level. Circumventing the ensemble requirement of molecule synchronisation, single-molecule studies are able to track the stochastic and heterogeneous kinetics of individual molecular machines. Here we establish a single-molecule assay to measure conformational change in the E. coli Hsp70 molecular chaperone DnaK. Re-arrangement of the nucleotide and substrate binding domains of DnaK present a useful system to follow large and dynamic changes in physical structure. Therapeutic agents targeted at eukaryotic Hsp70s to modulate activities in oncogenesis and neurodegenerative disorders, give real-world interest to understanding the basic mechanisms of DnaK. Using total internal reflection fluorescence (TIRF) microscopy we measure conformational cha nge in single DnaK molecules through Förster resonance energy transfer (FRET). Suitable constructs for the single-molecule FRET assay were selected from eight engineered DnaK cysteine variants. Biochemical activity assays showed variants retained wild type ability to undergo ATP-stimulated conformational change, hydrolysed ATP in the presence of co-chaperones grpE and DnaJ at 75-200% of wild type levels, and refolded denatured protein substrate at 20-35% the wild type level. Conjugation of these variants with thiol-reactive fluorophores gave proteins suitable for FRET assays, i.e. bearing both acceptor and donor fluorophores. Three DnaK variants, C15S/N254C/E430C, C15S/K321C/E430C, and C15S/N254C/R517C, showed nucleotide dependent changes to ensemble FRET indicative of conformational change. These three variants were used to establish a single-molecule FRET assay to observe transitions between discrete conformational states in immobilised and fluorescently labelled single DnaK proteins in real time. We show that DnaK visits multiple conformations and that nucleotide influences the distribution occupancies of these conformations. Rates of transitions between discrete conformations occur ≈ 100-2000 fold faster than ATP hydrolysis (0.7 x 10-3 s-1) in conditions without nucleotide. We survey a landscape of DnaK conformation transitions where structural states are visited dynamically, and used the calculated rates of transition to accurately predict occupancies observed in our data.