A General And Efficient Method For The Site-specific Dual-labeling Of Proteins For Single Molecule Fluorescence Resonance Energy Transfer

摘要

Single molecule fluorescence resonance energy transfer (sm-FRET) has emerged as a versatile tool for investigating biomolecular structure and dynamics. In contrast to ensemble measurements, direct measurements of conformational distributions and stochastic dynamics can be performed at single molecule resolution even in the context of complex structural landscapes and mixtures of molecular subpopulations. The strong distance dependence of energy transfer between donor (D) and acceptor (A) dyes makes smFRET especially useful for detailed studies of protein folding and conformational changes. However, a current inherent limitation is the need for site-specific labeling of the expressed protein with D and A dyes. Site-specific labeling is generally limited to the reaction of cysteine residues with various electrophiles; however, random labeling of two cysteine residues produces a mixture of two species (D-A + A-D) and poses significant problems for subsequent single molecule studies. This uncertainty can be problematic in the analysis of conformationally heterogeneous populations, can contribute to errors in distance measurements, and could mimic the existence of two biologically significant species. Hence, considerable interest has been placed on the design of alternate site-specific protein labeling strategies, to significantly expand the number and scope of proteins that can be investigated using smFRET.