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.

Conformational analysis of LYS(11-36), a peptide derived from the beta-sheet region of T4 lysozyme, in TFE and SDS.

The solution conformation of a peptide LYS(11-36), which corresponds to the beta-sheet region in T4 lysozyme, has been examined in aqueous solution, TFE, and SDS micelles by CD and 1H NMR spectroscopy. Secondary structure predictions suggest some beta-sheet and turn character in aqueous solution but predict a helical conformation in a more hydrophobic environment. The predictions were supported by the CD and NMR studies which showed the peptide to be relatively unstructured in aqueous solution, although there was some evidence of a beta-turn conformer which was maintained in 200 mM SDS and, to a lesser extent, in 50% TFE. The peptide was significantly helical in the presence of either 50% TFE or 200 mM SDS. TFE and SDS titrations showed that the peptide could form helical, sheet, or extended structure depending on the TFE or SDS concentration. The studies indicate that peptide environment is the determining factor in secondary structure adopted by LYS(11-36).

CD and NMR determination of the solution structure of a peptide corresponding to T4 lysozyme residues 38-51.

Solid phase methods have been used to synthesise a peptide corresponding to residues 38-51 of T4 lysozyme. The peptide, LYS(38-51), encompasses helix B in the crystal structure of T4 lysozyme. CD and 1H-NMR analysis showed that the peptide was unstructured in aqueous solution but adopted a helical conformation in the more hydrophobic environment provided by 50% TFE and SDS micelles. The solution structure derived from the NMR data was similar to that of the helix in the X-ray structure, although there was some fraying at the N-terminus.

Conformation of a peptide corresponding to T4 lysozyme residues 59-81 by NMR and CD spectroscopy.

The conformation, in solution, of a peptide corresponding to residues 59-81 from T4 lysozyme [LYS(59-81)] has been determined by 1H NMR and CD spectroscopy. This peptide spans the region corresponding to helix C in the crystal structure of T4 lysozyme. Secondary structure predictions indicated that the peptide would possibly be helical in an aqueous environment, but in a more hydrophobic environment the peptide would certainly adopt a helical conformation. This prediction was confirmed by the far-UV CD and NMR studies, which showed the peptide to be relatively unstructured in aqueous solution and significantly helical in the presence of either TFE or SDS micelles, although the 1H NMR results did give some indication of the presence of nascent helix in aqueous solution. For LYS(59-81), in TFE, the three-dimensional structure derived from the NMR data showed that the helix had a more pronounced curvature than the gradual bend observed in the crystal structure.