The synthesis of photolabile linkers and their applications in studies of protein folding

• Main Authors: Chih-Kai, Jacky Lin, 林至凱
• Other Authors: Sunney I. Chan
• Format: Others
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/99093673442904299281

碩士 === 國立清華大學 === 化學系 === 90 === The traditional stopped-flow methods for following the kinetic time course of protein folding reactions are limited by the dead-time of mixing. Moreover high concentrations of a denaturant are usually added to unfold proteins at the outset. In fact, how the denaturant affect folding pathways has been ignored. We cannot distinguish between the dissociation rate of denaturant and protein refolding rate. Using the photolabile linkers, a general method to monitor protein folding process shorter than 1 microsecond has been developed. The method is based on the photolysis of small organic protecting groups, or “cage” compounds. The compounds used in the studies are 3’,5’-dimethoxybenzoin (DMB) and bromoacetyl-carboxymethoxybenzoin (BrAc-CMB). These cage compounds are incorporated in to a three stranded β-sheet for the “sidechain” cage strategy and the N-terminal sequence of ubiquitin for “head-to-sidechain cyclization” strategy by Fmoc solid-phase peptide synthesis. DMB and Fmoc-Asp(DMB)-OH were synthesized by different reactions and conditions. The properties of Fmoc-Asp(DMB)-OH and the “sidechain” caged peptide were further studied by UV spectroscopy. The “sidechain” caged peptide has no clear structural difference from its wildtype according to the CD spectroscopy and 2D-NMR data. So, the caged position had to be changed. The “cyclization” strategy has more potential to unfold the peptide. Two cyclized peptides were synthesized and purified.