Raman optical activity characterization of native and molten globule states of equine lysozyme: Comparison with hen lysozyme and bovine α-lactalbumin
✍ Scribed by Ewan W. Blanch; Ludmilla A. Morozova-Roche; Lutz Hecht; Wim Noppe; Laurence D. Barron
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 2000
- Tongue
- English
- Weight
- 297 KB
- Volume
- 57
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine ␣-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22°C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at ϳ1340 cm Ϫ1 , which is assigned to a hydrated form of ␣ helix, is more similar to that in the ROA spectrum of bovine ␣-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calciumbinding ability of equine lysozyme and bovine ␣-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at ϳ1300 cm Ϫ1 in hen lysozyme that is assigned to an ␣ helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at ϳ1305 cm Ϫ1 , which may reflect greater heterogeneity in some ␣-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22°C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22°C and the apo form at pH 4.5 and 48°C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated ␣ helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine ␣-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at ϳ1551 cm Ϫ1 in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.