¹H‐NMR spectroscopy of bovine lens β‐crystallin

Abstract

'H-NMR spectroscopic studies of bovine eye lens P-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three P-crystallin aggregates are dominated by resonances from the pB2 subunit, particularly from the N-and C-terminal extensions of this subunit. Resonances from other subunits, which all have terminal extensions, are, in general, absent from spectra of the P-crystallin aggregates. Therefore, the @2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other a-crystallin subunits. Furthermore, resonances arising from the C-terminal extension of pB2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal extension of pB2 and the hydrophobic pBl subunit, which is only found in the octomer, is discussed. At higher temperatures (45OC) in the octomer, partial exposure of the C-terminal extension of pS2 occurs indicating that the octomer may be starting to break up into smaller aggregates.The light refractive power of the vertebrate eye lens results from the high concentration of soluble proteins, collectively referred to as crystallins. Some 40-48% of the crystallins in the bovine lens belong to the P-crystallin family [l, 21. The seven a-crystallin subunits have molecular masses ranging over 23-33 kDa [ 3 ] . As determined by size-exclusion chromatography, the P-crystallin subunits are aggregated species consisting of dimers, trimers and octomers, which are commonly referred to as pL2, pL, and PH respectively 14-61.Based on their sequence similarity with the y-crystallins, it was proposed that each P-crystallin subunit consisted of four 'Greek key' motifs folded into two domains with the Nterminal globular domain of all P subunits having extensions of various lengths [7, 81. In addition, it was postulated that the three basic P subunits, pSl, pS2 and pB3, have extensions from the C-terminal domains [7,9]. Unlike the globular domains which are highly conserved across species, the terminal extensions of the P subunits display considerable sequence variation across species, especially within the N-terminal extension [9]. It has been proposed that the N-and Cterminal extensions extend from the globular domains [8-101. In support of this proposal, limited proteolysis of the @32 dimer results in preferential cleavage in the terminal extensions leaving the globular domains intact [ll]. The function of the terminal extensions is currently unknown, but it has been speculated that they act as attachment sites for oligomer formation or for binding to other cellular components [9].

Age-related Changes in Human Lens Crystallins Identified by Two-dimensional Electrophoresis and Mass Spectrometry

“…However, the function of the Nterminal extensions of β-crystallins remains unknown. They may play a role in stabilization of β-crystallin oligomers, but this hypothesis is still under investigation (Cooper, Carver and Truscott, 1993 ;Kroone et al, 1994 ;Trinkl, Glockshuber and Jaenicke, 1994 ;Werten et al, 1996 ;Ajaz et al, 1997). The Nterminal extensions may also act as intermolecular spacers to keep crystallins in elongating lens fibers dispersed.…”

Sequence Analysis of βA3, βB3, and βA4 Crystallins Completes the Identification of the Major Proteins in Young Human Lens

“…The asparagine-proline sequence in ␤B2 occurs at the interface between the N-terminal extension and the first "Greek key" containing motif (22). 1 H NMR spectroscopy has confirmed that the Nterminal extensions of ␤-crystallins have relatively greater flexibility than the Greek key containing motifs (31). Therefore, the proteolytically resistant Asn 15 -Pro 16 region of ␤B2 may be relatively inaccessible compared to the Asn-Pro regions of ␤B1 and ␤A3 which are cleaved.…”

Deamidation of Human βB1 Alters the Elongated Structure of the Dimer

“…The long N-terminal extension of bB1 is rapidly cleaved and by 1 year of age, 50 % of bB1 within a human lens has lost 15 amino acids from the N-terminus (David et al, 1996;Lampi et al, 1998). Several studies have shown that b-crystallin extensions facilitate higher ordered associations (Cooper, Carver and Truscott, 1993;Ajaz et al, 1997;Werten et al, 1999) and that loss of the N-terminal extension of bB1 is correlated to a decrease in the bH aggregate (Ajaz et al, 1997;Lampi et al, 1998;Ma et al, 1998). Thus, modi®cations to bB1 are associated with changes in oligomer formation.…”