A New Method to Study the Early Kinetic Events in Protein Folding:Laser Flash Photolysis of Caged Peptides
Date Issued
2004
Date
2004
Author(s)
Huang, Jen-Tse
DOI
en-US
Abstract
Proteins do not perform their proper functions unless they can reach their native states. Studies of the early kinetic events play a key role because they provide a means to explore the beginning of the free energy landscape of protein folding. The aim of the project is to develop a new method to monitor the early kinetic events in protein folding (as fast as a few nanoseconds).
This methodology involves the deployment of a photolabile linker to “cage” a polypeptide chain to form a non-native state. In this thesis, we have developed a photolabile compound, Br-AcCMB, to serve as the cage. Taking advantage of the high quantum yield of this photolabile linker (ΦCh = 0.72), we have caged several different peptide models in their non-native states in order to address certain fundamental issues related to the formation of structural motifs during the early stages of protein folding. By the use of a pulse UV laser (~10-12 s), we can break the photolabile linker and monitor the refolding process by using photoacoustic calorimetry (PAC) or photothermal beam deflection (PBD). The time resolution is greatly improved by the use of PAC (from nanoseconds to a few microseconds) and PBD (from a few microseconds to several milliseconds).
We have synthesized various caged peptides including three b-sheets with different turn sequences (c-E12C, c-P6DE12C, and c-19merE11C), one b-hairpin (c-D6C), and two a-helices (c-VHPM12C and c-aE9C). By the use of nuclear magnetic resonance spectroscopy (NMR) and circular dichroism spectroscopy (CD), we have studied the structural alteration of these caged peptides before and after photolysis. Except for the b-hairpin model c-D6C that apparently has a slow folding rate, the folding kinetics of the other caged peptides all fall within the time resolution of PAC. After de-convolution, the time constant of the individual refolding process could be obtained. Temperature dependent PAC measurements were carried out in order to obtain certain thermodynamic parameters, including the volume change (ΔV), enthalpy change (ΔH), and kinetic parameters, including the activation energy (Ea). Kinetic measurements using PBD on the peptide c-D6C are still on going.
Because cyclization constraint limits the initial state of the caged peptide to a subset of conformational space, our approach does not allow us to monitor the global refolding. However, it has the distinct advantage of allowing one to study a system beginning from a well-defined state. Specifically, this method allows direct observation and comparison of the structural change of the peptides folding with different driving forces or nucleation centers (such as the nucleation at the turn or by hydrophobic interaction) from the same initial state in real time.
Subjects
光熱射折射儀
光聲波熱卡計
蛋白質
蛋白質摺疊
胜肽
photoacoustic calorimetry
pritein
photothermal beam deflection
protein folding
peptide
Type
thesis