A New Method to Study the Early Kinetic Events in Protein Folding : Laser Flash Photolysis of Caged Peptides
Pei-Yeh Chen1, Howard Jan1, Sheng-Fa Yu1, Hsian-Rong Tseng2, Tien-Yau Lu2, Wun-Shain Fann3, and Sunney I. Chan1
1Institute of Chemistry, Academia Sinica, Taipei, Taiwan, R. O. C.
2Department of Chemistry, National Taiwan University, Taipei, Taiwan, R. O. C.
3Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan, R. O. C.
Traditional stopped-flow techniques have been widely employed in the study of protein folding. Unfortunately, substantial refolding occurs in the dead-time of mixing, which is of the order of 1 ms. Recently, Chan and coworkers (ref.1) proposed a new method to monitor the early kinetic events in the protein folding at times as early as 1 nanoseconds. The approach is based on the development of photolabile linkers to "cage" proteins or peptide systems into covalent structures that differ from the native equilibrium structure of the protein. The application of a short laser pulse (~10-12 sec) is used to break the photolabile linker and initiate the refolding of the protein toward its native state. Since the photolabile linker could be broken in less than 10-10 sec, this strategy opens up a temporal window to allow the observation of the early kinetic events of the protein refolding process. Here, we successfully coupled the photolabile linker, BrAcCMB, to the N-terminus of the three-stranded b-sheet, E12C, and cyclized the peptide in a head-to-sidechain strategy by linking the thiol group of the cysteine residue to the N-terminal linker. CD and NMR experiments show that the cyclization of the peptide have altered the peptide structure substantially. Studies of the refolding kinetics following laser flash photolysis of the “denatured” peptide are in progress.
(Ref. 1) Hansen, K. C., Rock, R. S., Larsen, R. W. and Chan, S. I. (2000) J. Am. Chem. Soc., 122, 11567-11568.