A structural study of bovine lens α‐crystallin and its subunits by absorption and linear dichroism spectroscopy

Abstract

The structure of the bovine a-crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250-350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in acrystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of a-crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.One of the extraordinary features of the eye lens is its transparency despite an extremely high concentration of proteins (up to 35% by mass). This was assumed to be related to the spatial order and molecular structure of these proteins [l]. Thus, insight into this structure is important for an understanding of the clearness of the lens and aberrations to this, such as those occurring in some kinds of cataract. Structural information is most readily obtained from crystallographic X-ray diffraction. Unfortunately, one of the major lens proteins, a-crystallin, has, to date, resisted all attempts to crystallize it. Hence, direct structural information on this protein by X-ray diffraction is not available yet, and no consensus exists even about its rough structure [2, 31.Native a-crystallin is usually assumed to be an assembly of 30 -50 A-type and B-type subunits with a molecular mass of 20 kDa/subunit [4, 51. After separation and isolation of these subunits, they reaggregate in solution. In this way, pure aA and aB aggregates may be obtained, the molecular mass of these being smaller than that of native aggregates [5, 61. The primary structures of aA-crystallin and aB-crystallin show about 60% sequence similarity [7, 81. Hence, it has been assumed to be highly probable that the subunits are structurally related and fold into similar motifs [6, 9, 101. However, recently it was suggested, that there are slight differences both between the structure of reconstituted aA and aB aggregates [6] and between their stabilities [ll]. In order to obtain more structural information about a-crystallin and its subunits, we have studied them by ultraviolet absorption and linear dichroism (LD) spectroscopy.LD is the phenomenon that samples in which the molecules (or more precisely their transition dipole moments)Correspondence to M. Bloemendal,

Structure and Modifications of the Junior Chaperone α‐Crystallin

“…The in vivo situation is even more complex. The aggregate size of a-crystallin increases upon aging [32, 1421, a phenomenon that has been observed in v i m too [143]. Also aB-crystallin appears to form larger aggregates during ischemia of the heart [144], as is observed for small HSPs upon various forms of stress [34,35,371.…”

The isolation of lens crystallins using lens liquid as the solvent

“…The relatively high intensity of this peak in the chromatogram when measured at 307 nm might be due to oxidation (Borkman, Hibbard and Dillon. 1986;Bloemendal et al, 1989). In the latter case peak I might represent a weakly aggregated form of some unidentified compound that is unstable, when not in its native environment.…”