NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

Abstract The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times ( T 2 ) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P / P 0 =0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P / P 0 =1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo ( T 2 =3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T 2 =8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P / P 0 =1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T 2 of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.