Thermal stability of human α‐crystallins sensed by amide hydrogen exchange

Abstract

The ␣-crystallins, ␣A and ␣B, are major lens structural proteins with chaperone-like activity and sequence homology to small heat-shock proteins. As yet, their crystal structures have not been determined because of the large size and heterogeneity of the assemblies they form in solution. Because ␣-crystallin chaperone activity increases with temperature, understanding structural changes of ␣-crystallin as it is heated may help elucidate the mechanism of chaperone activity. Although a variety of techniques have been used to probe changes in heat-stressed ␣-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens ␣-crystallin using hydrogen/deuterium exchange in conjunction with enzymatic digestion and analysis by mass spectrometry. This technique has the advantage of sensing structural changes along much of the protein backbone and being able to detect changes specific to ␣A and ␣B in the native assembly. The reactivity of the amide linkages to hydrogen/ deuterium exchange was determined for 92% of the sequence of ␣A and 99% of ␣B. The behavior of ␣A and ␣B is remarkably similar. At low temperatures, there are regions at the beginning of the ␣-crystallin domains in both ␣A and ␣B that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of ␣A also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in ␣A 72-75 and ␣B 76-79, two regions considered critical for chaperone activity.

Small heat shock proteins: molecular structure and chaperone function

“…When considered in light of three-dimensional modeling, hydrogen exchange, has a loose exposed structure. This agrees with data obtained by use of chemical cross-linkers and mass spectrometry to examine bovine a-crystallin [112], but contrasts the idea that the amino-terminus is hidden internally due to its hydrophobicity [23,92,113,114]. Analysis of crystal structure indicates the 40 amino-terminal residues of wheat Hsp16.9 are housed within oligomer interiors, but they experience rapid hydrogen/deuterium exchange, suggesting the region undergoes conformational change and solvent exposure [24,86].…”

“…Truncation and other modifi cations reveal important features of the sHSP carboxy-terminal extension, a fl exible region enriched in polar and charged amino acid residues existing as a solvent-exposed random coil [35,96,100,103,113,[125][126][127][128] (fi g. 1). Removal of 11 residues from the carboxy-terminus of E. coli IbpB leads to dimer formation and loss of chaperone activity [100].…”

Small Heat-Shock Proteins: Paramedics of the Cell

“…Solid-state nuclear magnetic resonance spectroscopy (NMR) revealed that dimerisation of aB-crystallin mediated by b6 + 7 pertains also to full-length protein, but to date only one register, AP II , has been observed [54]. While relating hydrogen/deuterium exchange rates determined for full-length aB-crystallin [55] to the structure of the truncated dimer certainly reveals the interfaces to be dynamic, it remains to be elucidated to what extent multiple AP interfaces are populated in the oligomers at equilibrium in solution, and whether they interconvert. However, irrespective of these registry shifts, it is clear that, despite very similar basic monomer structures, two distinctly different modes of dimerisation have evolved across the kingdoms of life.…”

Heat perturbation of bovine eye lens α‐crystallin probed by covalently attached ratiometric fluorescent eye 4′‐diethylamino‐3‐hydroxyflavone

“…In the present work, we demonstrate that within the C-terminal domain of this protein (namely, in the environment of Cys-131 residue) temperature-induced structural changes occur too. Previously, according to the data obtained by hydrogen/deuterium exchange in conjunction with enzymatic digestion and mass spectrometry, the C-terminal domain of -crystallin was suggested to be involved in chaperone substrate binding 19,20 ; it was also observed that isolated C-terminal domains of -crystallin mostly preserve their chaperone-like activity. 38 It is known that the C-terminal domain is highly conservative among sHSPs, including -crystallin.…”

“…It was observed that subunit-subunit interactions and chaperone-like activity remain unaffected in spite of chemical modification of cysteine residues. 17 The C-terminal region of -crystallin subunits was shown to be important for its chaperonelike activity, [18][19][20] making this part of molecule interesting to study. Thus, a fluorescent label attached to the Cys residue may provide information about biologically important changes of the -crystallin structure.…”