ABSTRACT
We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.
Subject(s)
Peptide Fragments/chemistry , Peptides/chemistry , Protein Folding , Energy Transfer , Guanidine , Models, Molecular , Plant Proteins , Protein Conformation , Protein Denaturation , Serine Proteinase Inhibitors/chemistry , Spectrometry, Fluorescence/methodsABSTRACT
Protein synthesis by native chemical ligation has been an effective approach for the synthesis of proteins of moderate size. The utility of this approach for protein synthesis is demonstrated by the synthesis of a transcription factor, Zif 268 that contains three zinc finger domains. This synthesis highlights the modular nature of the chemical ligation approach and the ability to synthesize, handle and fold multiple domain proteins.
