Abstract
Proteins possess a complex and dynamic structure, which is influenced by external signals and may change as they perform their biological functions. We present an optical approach, distance-encoding photoinduced electron transfer (DEPET), capable of the simultaneous study of protein structure and function. An alternative to FRET-based methods, DEPET is based on the quenching of small conjugated fluorophores by photoinduced electron transfer: a reaction that requires contact of the excited fluorophore with a suitable electron donor. This property allows DEPET to exhibit exceptional spatial and temporal resolution capabilities in the range pertinent to protein conformational change. We report the first implementation of DEPET on human large-conductance K+ (BK) channels under voltage clamp. We describe conformational rearrangements underpinning BK channel sensitivity to electrical excitation, in conducting channels expressed in living cells. Finally, we validate DEPET in synthetic peptide length standards, to evaluate its accuracy in measuring sub- and near-nanometer intramolecular distances.
Highlights
Proteins possess a complex and dynamic structure, which is influenced by external signals and may change as they perform their biological functions
One limitation in particular, is that Förster resonance energy transfer (FRET) always provides distances between fluorescent donor and acceptor protein adjuncts. This is hardly a concern when determining inter-molecular distances in protein complexes; fluorescent adjunct distances diverge significantly from protein atom distances and orientations in the sub- and near-nanometer scale pertinent to protein structure and function. To address this and other limitations of FRET methods, we have developed a new optical approach based on an alternative mechanism of distancedependent modulation of fluorescence: distance-encoding photoinduced electron transfer, DEPET
PET-based Trp quenching of small fluorescent molecules involves brief collisional quenching and longer-lasting, static quenching, likely due to the formation of stable hydrophobic complexes[8,13]. Both mechanisms require van der Waals overlap between the Trp indole side-chain and the fluorescent moiety, i.e., contact; and both occur within the nanosecond time domain, rendering them indistinguishable in steady-state fluorescence measurements, such as those in DEPET
Summary
Proteins possess a complex and dynamic structure, which is influenced by external signals and may change as they perform their biological functions. An alternative to FRET-based methods, DEPET is based on the quenching of small conjugated fluorophores by photoinduced electron transfer: a reaction that requires contact of the excited fluorophore with a suitable electron donor This property allows DEPET to exhibit exceptional spatial and temporal resolution capabilities in the range pertinent to protein conformational change. This is hardly a concern when determining inter-molecular distances in protein complexes; fluorescent adjunct distances diverge significantly from protein atom distances and orientations in the sub- and near-nanometer scale pertinent to protein structure and function To address this and other limitations of FRET methods, we have developed a new optical approach based on an alternative mechanism of distancedependent modulation of fluorescence: distance-encoding photoinduced electron transfer, DEPET. The distance between the Cys Cα and the Trp Cβ atoms can be determined: this information is useful for evaluating the orientation of the Trp side-chain with respect to the fluorescently-labeled site
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
