INFRARED AND CIRCULAR-DICHROISM SPECTROSCOPIC CHARACTERIZATION OF STRUCTURAL DIFFERENCES BETWEEN BETA-LACTOGLOBULIN-A AND BETA-LACTOGLOBULIN-B

Structural differences between two genetic variants of bovine beta-lac toglobulins (type A and B) in aqueous solutions were characterized usi ng Fourier transform infrared and circular dichroism spectroscopies. T o probe differences in structural dynamics, the effects hydrogen-deute rium exchange were also compared for the two proteins. The infrared sp ectra recorded in H2O solution for the two proteins were nearly identi cal in the conformationlly sensitive amide I region. The only exceptio ns were small differences at the band ascribed to a high-wavenumber be ta-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 solut ion, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent w ith the structural differences of the two variants at amino acid resid ues 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 spec tra for the two proteins were essentially identical, both before and a fter hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchan ge 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 prot eins. Since the rate of exchange was greater for variant A, it was con cluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectrosco py and hydrogen-deuterium exchange has great potential in characteriza tion of even subtle structural differences in proteins induced by natu rally occurring point mutations and/or site-directed mutagenesis.