The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate.
T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.
Kato
Hidenori
H
Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, Building 37, Room 6114E, Bethesda, MD 20892, USA.
Vu
Ngoc Diep
ND
Feng
Hanqiao
H
Zhou
Zheng
Z
Bai
Yawen
Y
D017122:Bacteriophage T4 / Q000201:enzymology*; D002942:Circular Dichroism; D005453:Fluorescence; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors
D050505:Mutant Proteins; D006859:Hydrogen; D009113:Muramidase