New fluorescence methods for protein folding and conformational studies

Fluorescence spectroscopy has been extensively used in the study of protein folding, dynamics and function. Fluorescence resonance energy transfer (FRET), in particular, has proven to be very useful in this regard. However, FRET studies commonly employ exogenous fluorescent dyes, which may cause undesirable consequences, such as those due to nonspecific labeling or the bulky size of the probes. Amino acid fluorophores, therefore, provide an attractive alternative for FRET applications as they are smaller and easy to incorporate into the protein of interest. In the first part of this thesis, we explore the applicability of an amino acid FRET pair, p-cyanophenylalanine (Phe CN) and tryptophan (Trp), to the study of protein folding using two model protein systems. We demonstrate that quantitative information regarding the folding/unfolding process can be extracted from a single FRET titration experiment using a theoretical method we developed. Subsequently, we show that this FRET pair can be used in conjunction with a stopped-flow technique to directly dissect the binding and folding kinetics of intrinsically disordered proteins. Building on this work, we show that other non-natural amino acids, such as 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW), can also be used as FRET acceptors to PheCN, each with specific advantages over Trp. Finally, we demonstrate that PheCN, Trp and 7AW constitute a multi-FRET system that can be used to simultaneously monitor relative movements between different regions of a protein, thus allowing additional structural information to be extracted from a single FRET experiment. In the second part of this thesis, we expand the utility of fluorescence correlation spectroscopy (FCS) to the study of membrane proteins. We illustrate, using the Influenza A M2 proton channel as a model, how fluorescence fluctuations that arise from a single extrinsic fluorophore (from both a photoinduced electron transfer process and motions of the fluorophore) can be used to probe the dynamics and/or conformational distributions of proteins embedded in a membrane environment