Zinc Porphyrin as a Donor for FRET in Zn(II)cytochrome c

摘要

Fluorescence resonance energy transfer (FRET) is a powerfulntechnique for the study of the structure and function of biomoleculesnin that it allows for the direct measurement of distances on thennanometer scale. FRET is particularly useful for applications suchnas single-molecule (SM) spectroscopy,n1nand the need for fluores-ncently labeled molecules for ensemble and SM FRET has stimulatednthe development of new techniques for site-specific labeling ofnproteins,n2nas well as improvements to individual fluorophorenphotostability.n3nEven with these innovations, it is still difficult tonsite-specifically label a single amino acid chain with two fluoro-nphores suitable for FRET.n2,4nRecently, it was demonstrated that anzinc porphyrin (ZnP) cofactor in Zn(II)-substituted horse heartncytochrome c (Zn cyt c) can serve as a FRET acceptor in proteinnfolding studies.n5nSince ZnP is an intrinsic fluorophore, FRET studiesnof Zn cyt c require attachment of only one dye molecule, simplifyingnthe labeling procedure while minimizing perturbation to the proteinnstructure. However, the applicability of ZnP as an acceptor for SMnFRET studies is inherently limited by the low fluorescence quantumnyield (QY) of the cofactor.n5nHere we show that ZnP is an efficientnFRET donor to an Alexa 660 (A660) dye acceptor in Zn cyt c.nThe large separation of donor (590 nm) and acceptor (690 nm)nfluorescence energies allows for a simplified analysis of acceptor-nsensitized emission, which results in more accurate FRET efficiencynvalues than reported previously.