Degree Name

Doctor of Philosophy (PhD)


Department of Chemistry and Department of Biological Sciences - Faculty of Science


Alpha-crystallin is a member of the small heat shock protein (sHsp) family which exists as a multimer of alphaA- and alphaB-crystallin subunits in the ratio of 3:1 in the lens, where it was first identified. It is an intracellular molecular chaperone, capable of interacting with a multitude of target proteins to prevent their aggregation and precipitation. Initially considered to be solely a lens protein, individual alphaA- and alphaB-crystallin proteins have since been found in other organs with alphaB-crystallin in particular appearing to play a role in many neurodegenerative disorders (e.g. Alzheimers, Parkinsons and Creutzfeldt-Jakob diseases). Due to the dynamic nature of alpha-crystallin oligomers and the propensity for subunit exchange, crystallisation of the protein has been impossible. As a result, the mechanisms by which alpha-crystallin functions remain elusive, as does a complete picture of the chaperones quaternary structure. In this study, recombinant human alphaA- and alphaB-crystallin were expressed and purified using conventional methods (Horwitz, J., Huang, Q-L., Ding, L. and Bova, M. P. (1998) Methods in Enzymology 290:363-383). A series of mutants of alphaA- and alphaB-crystallin were also constructed, with mutation sites concentrated in the C-terminal region of the protein and in particular the solvent-exposed and flexible C-terminal extension. This extension, which comprises 10 and 12 amino acids in human alphaA- and alphaB-crystallin, respectively, behaves in a similar manner to an unstructured peptide in solution. Previous NMR spectroscopic studies have indicated that it serves a crucial role in binding target proteins. C-Terminal extension mutants K175L, K174A/K175A, E164A/E165A, I159A/I161A and R163STOP (alphaB-crystallin) and S172L, T168L and R163STOP mutants (alphaA-crystallin) were produced and purified in the same manner as for the wildtype proteins. The expected masses of mutants were confirmed by electrospray mass spectrometry (ESI-MS). Complete purification, however, of S172L and R163STOP alphaA-crystallin was not achieved due to decreased aggregate size and excessive hydrophobicity, respectively. Purified wildtype and mutant proteins were structurally and functionally characterised using a variety of spectroscopic techniques. These included chaperone assays under both reduction and heat stress with insulin and beta(subscript L)-crystallin as target proteins, respectively, intrinsic tryptophan fluorescence spectroscopy, far- and near-UV circular dichroism (CD) spectroscopy, thermostability studies, size-exclusion high-performance liquid chromatography (HPLC), mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Many of the alphaB-crystallin mutants purified successfully and provided insight into specific amino acid residues that are important for the chaperone action of the protein. These include the I-X-I motif at the C-terminal end of the protein which is highly conserved throughout sHsps and is thought to be critical for oligomeric assembly. Mutation of both isoleucine residues in the alphaB-crystallin I-X-I motif to alanine resulted in a protein which formed larger oligomeric complexes than the wildtype protein. Truncation of the C-terminal extension of alphaB-crystallin resulted in a protein with severely impaired chaperone ability, increased tendency to aggregate and disrupted secondary, tertiary and quaternary structures. These results suggest that the polar and flexible C-terminal extension is also necessary for uniform oligomeric assembly as well as for the solubility of ?-crystallin as a whole. Chaperone and thermostability studies on the double glutamic acid mutant (E164A/E165A) showed that these highly charged residues are critical to the solubility of alphaB-crystallin at higher temperature. Consistent with this, ion-pairs and the formation of salt bridges between charged amino acids on the surfaces of thermophilic proteins are thought to be responsible for their increased thermostability. Recombinant human alphaA- and alphaB-crystallin were uniformly (superscript 15)N-labelled for the purposes of 2D Nuclear Magnetic Resonance spectroscopy (NMR) studies. Measurement of (superscript 15)N relaxation time constants (T[subscript 1] and T[subscript 2]) and (superscript 15)N Nuclear Overhauser Effects (NOEs) for both wildtype proteins and the K175L and I159/I161A mutants of alphaB-crystallin have provided detailed information on the relative flexibilities of residues in the proteins C-terminal extension. Substitution of a leucine residue for the C-terminal lysine (K175) increased extreme C-terminal mobility and substitution of the isoleucine pair of the I-X-I motif with alanine residues led to a disruption of flexibility throughout the C-terminal extension. (superscript 15)N T(subscript 1) and T(subscript 2) and (superscript 15)N NOE values were also determined for (superscript 15)N-labelled alphaA-crystallin in the presence of reduced alpha-lactalbumin in order to gain information on changes in the flexibility of the C-terminal extension upon chaperone interaction with a stressed target protein. Upon formation of a chaperone-target protein complex, the flexibilities of C-terminal residues of alphaA-crystallin were equalised across the extension indicating that the entire extension was involved in interaction with the target protein to some extent. The R120G alphaB-crystallin mutant, which is associated with desmin-related myopathy and cataract in humans was also expressed and purified for the purposes of further structural characterisation. Previous studies on this mutant have provided some ambiguous results with regard to its chaperone ability and general structural stability. It was found that in addition to being intrinsically unstable and susceptible to unfolding, R120G alphaB-crystallin underwent C-terminal proteolysis with time. Furthermore, R120G alphaB-crystallin exhibited marked substrate specificity and in fact, acted as an anti-chaperone in the presence of reduced ?-lactalbumin. Under these conditions, R120G alphaB-crystallin promoted the aggregation of the molten globule state of alpha-lactalbumin and co-precipitated with it out of solution. This study, therefore provided several insights into structural and functional aspects of alpha-crystallin small heat shock chaperone proteins. [Note: this abstract contained scientific formulae that would not come across on this form. Please see the 01Front files abstract for the full details.]

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