Stabilization of protein by replacement of a fluctuating loop: structural analysis of a chimera of bovine alpha-lactalbumin and equine lysozyme.
Equine lysozyme is a calcium-binding lysozyme and an evolutional intermediate between non-calcium binding c-type lysozyme and alpha-lactalbumin. We constructed a chimeric protein by substituting the fluctuating loop of bovine alpha-lactalbumin with the D-helix of equine lysozyme. The substitution affects the protection factors not only in the fluctuating loop but also in the antiparallel beta-sheet, the A- and B-helices, and the loop between the B-helix and the beta-sheet. Amide protons in these regions of the chimera are more protected from exchange than are those of bovine alpha-lactalbumin. We used model-free analysis based on 15N nuclear magnetic resonance relaxation measurements to investigate the dynamics of the main chain of the chimera and showed that the fluctuating loop of the chimera is as rigid as three major helices. When we analyzed the chemical shift deviations and backbone HN-H(alpha) scalar coupling constants, we found that the chimera showed an alpha-helical tendency in residues around the fluctuating loop. Our results suggest that the replacement of a highly fluctuating loop in a protein with a rigid structural element in a homologous one may be useful to stabilize the protein structure.
Tada
Masahito
M
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan.
Kobashigawa
Yoshihiro
Y
Mizuguchi
Mineyuki
M
Miura
Kazunori
K
Kouno
Takahide
T
Kumaki
Yasuhiro
Y
Demura
Makoto
M
Nitta
Katsutoshi
K
Kawano
Keiichi
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004795:Enzyme Stability; D005456:Fluorescent Dyes / Q000378:metabolism; D006736:Horses; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D007768:Lactalbumin; D009113:Muramidase