Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B.
Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high-wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.
Dong
A
A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Matsuura
J
J
Allison
S D
SD
Chrisman
E
E
Manning
M C
MC
Carpenter
J F
JF
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D017666:Deuterium Oxide; D007782:Lactoglobulins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water
D007782:Lactoglobulins; D012996:Solutions; D014867:Water; D017666:Deuterium Oxide