| The field of protein folding is considered to be one of the frontiers in biophysics and structural biology. The helix-coil transition of the polypeptide is considered to be one of the simplest protein folding puzzles. Despite its simplicity, the helix formation mechanism and time scale are still under investigation. Early experimental and theoretical studies suggest that the helix-coil transition proceeds on sub-microsecond time scale. Surprisingly, a stopped-flow CD study reveals that the helix-coil transition may occur on a much slower time scale, i.e. millisecond. Therefore, a rather complete study of the subject is needed. Here, a systematic study of the polypeptide helix-coil transition kinetics of two model α-helical peptides and the isotopic labeled derivatives of one of the peptides is presented. In order to study the fast kinetics, fast initiation and detection methods have to be used. Our general approach to study the fast events in protein folding is to use laser-induced temperature jump (T-jump) technique to initiate refolding or unfolding events on nanosecond timescale, and use time-resolved infrared (IR) spectroscopy to probe the subsequent folding kinetics and structural ordering along the folding pathways.; Infrared spectroscopy is one of the extensively used techniques in protein conformational studies. The amide I band is a well-established conformational reporter. It has been used extensively to extract information regarding the secondary structure composition in proteins. The major disadvantage of using amide I band, however, is that it cannot report on local environment. During many biological processes, the environment of amino acid side-chains will undergo significant change. So, it is quite advantageous to introduce a local environment probe that can report the side-chain surroundings. Here, the C≡N group attached to the end of the amino acid side-chain is proposed to serve as a local environment probe. By coupling with fast initiation methods, the nitrile-derivatized amino acids can potentially be used to study a variety of structural and dynamic questions, such as side-chain packing, side-chain-side-chain interaction, peptide-protein binding, and protein-protein interaction. |
