The folding pathway of T4 lysozyme: the high-resolution structure and folding of a hidden intermediate.
Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rd-apocytochrome b(562) and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the N-terminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multi-dimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many non-native side-chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins.
Kato
Hidenori
H
Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, Building 37, Room 6114E, Bethesda, MD 20892, USA.
Feng
Hanqiao
H
Bai
Yawen
Y
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D019143:Evolution, Molecular; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics
D050505:Mutant Proteins; D006859:Hydrogen; D009113:Muramidase