Lysozyme Maintains Most of its Helical Conformation in Its Early Oligomeric State: Molecular Insight by Raman Spectroscopy
Posted: 3 Feb 2020
Date Written: January 30, 2020
We report here both the secondary and tertiary structure intricacy of highly folded hen egg-white lysozyme (HEWL) in its crystalline state, weakly folded oligomer assembly and steric zipped thermodynamically most stable fibrillar structure by Raman spectroscopic method. The conformation marker Raman amide I band in wet crystals clearly appeared at 1656 cm−1 indicating a major α-helical structure of the crystalline protein at both the pHs (pH 7.4 and pH 4.8) conditions. Freshly prepared protein solution at pH 1.6 also preserved most of its α helical fold and the amide I band appeared at 1656 cm-1. In addition, the presence of a distinct Raman band at ~932 cm-1, a well-defined marker for α‐helical fold linked to N-Cα-C stretching, in crystals and monomeric protein in solution clearly suggested the presence of a helical component at very low pH. Incubation of the protein at low pH and elevated temperature (60 oC) produced nanosize oligomer assembly. A substantial Raman intensity at 1660 cm-1 and the N Cα C skeletal a band at 930 cm-1 indicated the presence of a significant amount of α-helical folds in the oligomeric assembly state. A band fitting analysis revealed ~40% α-helical secondary structure, which was ~5 % less than the monomeric protein samples. Raman difference spectrum evidenced certain changes in α-helicity and a small increase of β-sheet and PPII like secondary structure in the oligomeric state of the protein. Compared to monomeric and oligomeric sample, a substantial amount of loss in disulfide (-S-S-) linkages were observed and the Raman band at 505 cm-1 decreased in the fibrillar state. In the fibrillar state, α-helical structure decreased substantially, amide I band became narrower and appeared at 1671 cm 1. Most of the Raman bands of aromatic residues broadened in the oligomeric state. The higher energy component band of tryptophan Fermi doublet at 1360 cm 1 was enhanced in the fibrillar state compared to its oligomeric condition and indicated that tryptophan microenvironment became more hydrophobic in the fibrillar state. The ~875 cm-1 Raman bands shifted to high energy side and became sharper in the fibrillar state. The Fermi doublets (~830 /~855 cm-1) of the tyrosine residues were much distinct in matured fibrils. The intensity ratio (I855 /I830) was ~0.9 for fibrils and for oligomers the value was ~1.5. It suggested that the tyrosyl hydroxyl in the oligomeric state act both as H-bond donor and acceptor, however in the fibrillar condition no of tyrosine residues involved in H-bond donation may be increased. Thus some of the tyrosine and tryptophan residues may be in hydrophobic zipper conformation with less fluctuation compared to the protein in its oligomeric state with molten globule like partially unfolded tertiary architecture rich with PPII like secondary structure.
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