Abstract
How do proteins fold? Scientists still can’t find a rule to explain how it works. Proteins organize themselves into specific three-dimensional structures through the myriads of conformational changes. In order to understand the intrinsic principle of protein folding, early events of folding process have to be systematically explored. Here we proposed a photo-triggered caging-strategy to analyze the whole folding process of a protein on a nanosecond time-scale. Our target protein is an antifreeze protein, RD1, taking advantage of the existence of a small cavity in the center of the protein. We change Ala-7 of RD1 to Cys (designated RD1-A7C) by site-directed mutagenesis and add a photolabile cage group to the residue Cys around the cavity. The bulky size of the cage can hinder the hydrophobic packing and unfold the protein. A short laser pulse is used to break the photolabile cage and to initiate the refolding of the protein toward its native state. In this work, we synthesized the photolabile cage compound 4-hydroxyphenacyl bromide (HPB). The protein RD1-A7C was expressed and purified. HPB was successfully added to the RD1-A7C protein (denoted RD1-A7C-HP). Finally, the photolysis, structural characterization, and folding kinetics of RD1-A7C-HP were studied.