Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments.
Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.
Najbar
L V
LV
Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.
Craik
D J
DJ
Wade
J D
JD
McLeish
M J
MJ
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D002942:Circular Dichroism; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000138:chemical synthesis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010442:Peptide Chain Initiation, Translational; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013501:Surface-Active Agents; D014270:Trifluoroethanol / Q000737:chemistry
D008823:Micelles; D010446:Peptide Fragments; D013501:Surface-Active Agents; C108356:T4 lysozyme (11-36); C094995:T4 lysozyme (38-51); D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D009113:Muramidase