Probing α-crystallin structure using chemical cross-linkers and mass spectrometry

Native α-crystallin [1], by far the most abundant protein found in the lens, is a hetero-oligomeric polydisperse complex about 700-800 kDa in size [2]. The oligomer is composed of about 35-40 noncovalent subunits in a 3:1 mixture of two highly homologous 20 kDa proteins, αA-crystallin and αB-crystallin, respectively. Tertiary structure is thought to be similar to that of other small heat shock proteins (sHSPs) where the crystal structure has been solved [3,4]. For example, the “β sandwich fold” of the region collectively known as the “highly conserved α-crystallin domain” is thought to be present in all sHSPs [1]. The need for alternative techniques to explore the tertiary and dynamic quaternary structure of α-crystallin is acute. Once thought to have only structural and optical functions in the lens, αB-crystallin is a molecular chaperone [5] and has been found in many other tissues [6,7]. Moreover, the role of αBcrystallin in several neurological pathologies in addition to cataract, including Alzheimer’s disease [8], and recent findings that crystallins undergo fibril formation characteristic of amyloid pathology [9,10] argue for the accelerated development and application of new techniques to study both native and disease states of this important protein. The polydisperse nature of α-crystallin is thought to have been the main obstacle to acquisition of high resolution X-ray crystallograghic data to determine its structure [1]. Over many years several models for the tertiary and quaternary structure of α-crystallin have been proposed using a variety of analytical techniques [11-18]. Many sought to reconcile the amphiphilic and hetero-oligomeric nature of α-crystallin with both its optical and molecular chaperone properties. Wistow ©2004 Molecular Vision