Studying submicrosecond protein folding kinetics using a photolabile caging strategy and time-resolved photoacoustic calorimetry.
Kinetic measurement of protein folding is limited by the method used to trigger folding. Traditional methods, such as stopped flow, have a long mixing dead time and cannot be used to monitor fast folding processes. Here, we report a compound, 4-(bromomethyl)-6,7-dimethoxycoumarin, that can be used as a "photolabile cage" to study the early stages of protein folding. The folding process of a protein, RD1, including kinetics, enthalpy, and volume change, was studied by the combined use of a phototriggered caging strategy and time-resolved photoacoustic calorimetry. The cage caused unfolding of the photolabile protein, and then a pulse UV laser (∼10(-9) s) was used to break the cage, leaving the protein free to refold and allowing the resolving of two folding events on a nanosecond time scale. This strategy is especially good for monitoring fast folding proteins that cannot be studied by traditional methods.
Chen
Hsin-Liang
HL
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Hsu
Jack C-C
JC
Viet
Man Hoang
MH
Li
Mai Suan
MS
Hu
Chin-Kun
CK
Liu
Chia-Hsun
CH
Luh
Frederick Y
FY
Chen
Silvia S-W
SS
Chang
Evan S-H
ES
Wang
Andrew H-J
AH
Hsu
Min-Feng
MF
Fann
Wunshain
W
Chen
Rita P-Y
RP
D000595:Amino Acid Sequence; D021343:Antifreeze Proteins, Type III / Q000737:chemistry*; D002151:Calorimetry; D002942:Circular Dichroism; D003198:Computer Simulation; D003374:Coumarins / Q000737:chemistry*; D007700:Kinetics; D007834:Lasers; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D021343:Antifreeze Proteins, Type III; D003374:Coumarins; C092813:RD3 protein, eel; C053769:4-bromomethyl-6,7-dimethoxycoumarin