Conformational changes during apoplastocyanin folding observed by photocleavable modification and transient grating.
A new method to investigate the initial protein folding dynamics is developed based on a pulsed laser light triggering method and a unique transient grating method. The side chain of the cysteine residue of apoplastocyanin (apoPC) was site-specifically modified with a 4,5-dimethoxy-2-nitrobenzyl derivative, where the CD and 2D NMR spectra showed that the modified apoPC was unfolded. The substituent was cleaved with a rate of about 400 ns by photoirradiation, which was monitored by the disappearance of the absorption band at 355 nm and the increase in the transient grating signal. After a sufficient time from the photocleavage reaction, the CD and NMR spectra showed that the native beta-sheet structure was recovered. Protein folding dynamics was monitored in the time domain with the transient grating method from a viewpoint of the molecular volume change and the diffusion coefficient, both of which reflect the global structural change, including the protein-water interaction. The observed volume decrease of apoPC with a time scale of 270 micros is ascribed to the initial hydrophobic collapse. The increase in the diffusion coefficient (23 ms) is considered to indicate a change from an intermolecular to an intramolecular hydrogen bonding network. The initial folding process of apoPC is discussed based on these observations.
Hirota
Shun
S
Department of Physical Chemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. hirota@mb.kyoto-phu.ac.jp
Fujimoto
Yukari
Y
Choi
Jungkwon
J
Baden
Naoki
N
Katagiri
Noriko
N
Akiyama
Masako
M
Hulsker
Rinske
R
Ubbink
Marcellus
M
Okajima
Toshihide
T
Takabe
Teruhiro
T
Funasaki
Noriaki
N
Watanabe
Yoshihito
Y
Terazima
Masahide
M
D001059:Apoproteins / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004058:Diffusion; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007834:Lasers; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D009578:Nitrobenzenes / Q000737:chemistry; D010970:Plastocyanin / Q000737:chemistry*; D011487:Protein Conformation / Q000528:radiation effects*; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000528:radiation effects*; D014867:Water / Q000737:chemistry
D001059:Apoproteins; D009578:Nitrobenzenes; C040778:apoplastocyanin; D014867:Water; D010970:Plastocyanin; D003545:Cysteine