Development of new strategies towards site-specific fluorescent labeling of proteins for use in sensitive biophysical studies

Fluorescence spectroscopy is one of the most sensitive and versatile tools available in biochemical research. Incorporation of organic fluorophores into protein sequences provides a powerful strategy to investigate structure/function relationships on selected proteins within heterogeneous environments. The success of these studies relies on the ability to introduce fluorescent reporter groups into predetermined sites within the protein sequence with both high efficiency and fidelity. This thesis explores two methods of site-specific labeling, chemical and biosynthetic.;Biosynthetic incorporation of commercial fluorophores and novel biotinylated fluorophores into protein sequences. Introduction of N-acyl derivatives of methionyl-tRNAfmet into a cell-free, coupled transcription/translation system provides a reliable method for site-specific labeling of proteins. Fluorescent dyes including Cy3, Cy5 and BODIPY-FL as well as a biotin affinity tag were introduced into a variety of protein sequences using this method. Also, three novel bi-functional labels were constructed through the combination of either a Cy5 or BODIPY-FL fluorophore with a biotin affinity tag and made amine-reactive upon further derivatization to NHS or NHSS esters. Although incorporation of these bi-functional reagents was possible, increasing the size and structural complexity of the N-acyl derivatives reduced the ability of these tRNAs to initiate protein synthesis. In Addition, a purification strategy was developed using anti-cyanine antibodies to isolate Cy3- and Cy5-labeled proteins from the crude translation mixture. Finally, a quantitative, high-throughput assay to screen for protein-protein interactions based on this N-terminal fluorescence labeling strategy is proposed and initial progress towards achieving this goal is reported.;Chemical incorporation of fluorescent dyes into the ion channel gramicidin. A variety of commercially available fluorophores were incorporated at the C-terminus of the peptide ion channel gramicidin through the use of semi-synthetic peptide chemistry. Combined optical and electrical studies were performed at the single-molecule level with these fluorescent gramicidins. Heterodimer channels consisting of one Cy3-labeled peptide and one Cy5-labeled peptide were identified optically by the appearance of a FRET signal and were correlated with the electrical signal from the same channel. This result constitutes the first example of simultaneous optical and electrical correlation of the gating event of an ion channel measured at the single-molecule level.