Probing the Fast Folding Kinetics:Folding Studies of an Antifreeze Protein RD1 by Using Photolabile Caging Strategy and Laser Flash Photolysis
Date Issued
2009
Date
2009
Author(s)
Hsu, Chun-Chieh
Abstract
In order to understand the intrinsic principle of protein folding, events of the fold-ing process have to be systematically explored. In this work, the folding information of a small protein (RD1, about 7 kDa) including kinetic, enthalpy and volume change were reported by combining the photo-triggered caging-strategy and time-resolved photo-acoustic calorimetry. This strategy required the mutation with Ala-7 to Cys (designated RD1-A7C) that was introduced to incorporate a photolabile cage group, 4-(bromomethyl)-6,7-dimethoxycoumarin, to unfold the protein. The structural proper-ties of the caged protein were analyzed by nuclear magnetic resonance spectroscopy (NMR) and circular dichroism spectroscopy (CD). A pulse UV laser (~10-9 s) was used to break the photolabile cage and two events were observed in the refolding of RD1-A7C toward its native state by using photoacoustic calorimetry (PAC). The fast event, which has a folding time of 20 ns and a volume change of -9.7 mL/mol, was ex-plained as the result of rapid volume contraction. This event was followed by a con-formational rearrangement, which has a folding time ~ 470 ns and small volume change (-1.4 mL/mol).
Subjects
folding
cage
photolabile
photoacoustic calorimetry
kinetics
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