Pre- heteronuclear biomolecular NMR works

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Article [1] presents nearly complete ¹H assignment of 129 residue hen egg lysozyme based on Phase-sensitive 2D NOESY, Phase-sensitive COSY and RELAY and Double RELAY spectra culminating a 15 year effort of assigning lysozyme exclusively by ¹H NMR. The article presents a detailed illustrated explanation of the sequential assignment process. Spectra were recorded at 35^oC. Samples were 7 mM, pH=3.8. Total of four samples were analyzed:

1. in 90% H₂O/10% D₂O,
2. fully D₂O exchanged sample repared by heating at 80^oC for 10 min
3. partially D₂O exchanged sample prepared by dissolving protein in D₂O at 35^oC; this sample retained slowly exchanging amide protons
4. reverse-exchanged sample prepared by dissolving lyophylized fully D₂O exchanged sample in 80% H₂O/20% D₂O. Solid sample was first dissolved in D₂O then a proportional amount of H₂O was added; this sample was used to detect quickly exchanging amide protons

References

1. Redfield, C and Dobson, CM. Sequential proton NMR assignments and secondary structure of hen egg white lysozyme in solution. Biochemistry 27(1):122--136, 1988. BibTeX [red98]