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Abstract
Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.
Highlights
Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli
photoinduced electron transfer (PET) fluorescence reporters for protein conformational change can be designed by placing fluorophore and Trp on a protein alongside the targeted reaction coordinate[15,16,17]
The PET reporters are designed to probe three elementary, ATPase-driven structural changes of the chaperone, namely closure of the “ATP-lid” over the nucleotide binding pocket in the N-terminal domain (NTD) (Lid), swapping of the N-terminal βstrands associated with inter-subunit dimerization of NTDs and intra-subunit association of NTD and middle domain (MD) (NM-association) (Fig. 1a)
Summary
Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. We present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. PET harbours the potential to simultaneously probe multiple conformational changes within a single protein molecule using one fluorescence colour per coordinate measured on a multicolour setup. Understanding Hsp90’s chaperone mechanism could form the basis of new therapeutic approaches
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