Contributions of aromatic pairs of human Gamma-D-Crystallin to its folding, stability, aggregation, and interaction with human Alpha B-Crystallin

Abstract

Two distinct groups of proteins, a-crystallins and [Beta][gamma]-crystallins, constitute 90% of the vertebrate eye lens soluble proteins. Long-term solubility and stability against unfolding and aggregation are essential properties of crystallins and crucial to the function of the lens. Aggregation of crystallins in the lens causes light scattering and directly contributes to development of cataract, the leading cause of blindness in the world. The amino acid determinants of these biochemical/biophysical properties of crystallins are not entirely understood. Aromatic residues in proteins have been shown to be important determinants of their folding pathways, native-state stability, aggregation propensity and other intermolecular interactions. In this thesis study, I have investigated the contributions of the paired aromatic residues of human yD-crystallin (H[gamma]D-Crys) to its folding, stability, aggregation, and interaction with the chaperone human aB-crystallin (H[alpha][Beta]-Crys). H[gamma]D-Crys is a highly stable protein that remains folded in the nucleus of the eye lens for the majority of an individual's lifetime. Like other [Beta][gamma]-crystallins, H[gamma]D-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs and eight [Beta]-strands. Six conserved aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in H[gamma]D-Crys. Four among them are located at conserved [Beta]-hairpins of the Greek key motifs, thus termed "Greek key pairs". The Greek key pairs have the consensus sequence Y/FXXXXY/FXG and are one of the defining features of the [Beta][[gamma]-crystallin family. Ultraviolet (UV) damage to these aromatic residues in [Beta][gamma]-crystallins may contribute to unfolding and aggregation of the proteins, leading to development of cataract. [Alpha]-Crystallins belong to the small heat shock protein (sHsp) family and have both structural and chaperone functions in the lens. Human a-crystallins form polydisperse oligomers of 15-60 subunits, with aA:aB ratio about 3:1 in vivo. The core a-crystallin domain (aCD) of acrystallins has an immunoglobulin (Ig)-like p-sandwich fold, but the quaternary structure of acrystallin remains to be fully solved. Like other sHsps, a-crystallins exert their chaperone function by sequestering partially-unfolded and aggregation-prone substrates in an ATPindependent manner, thus preventing their aggregation. The chaperone-substrate interactions of a-crystallins and other sHsps remain poorly understood. To investigate the roles of the paired aromatic residues in H[gamma]D-Crys, mutant proteins with these aromatic residues substituted with alanines were constructed and expressed in E. coli. All mutant proteins maintained native-like secondary structures by circular dichroism (CD). Except F 115A and F 117A, all mutant proteins had lower thermal stability than the wildtype (WT) protein. Equilibrium unfolding/refolding experiments in guanidine hydrochloride (GuHCl) showed that all mutant proteins had lower thermodynamic stability than the WT protein. Nterminal domain (N-td) substitutions shifted the N-td transitions to lower GuHCl concentrations, but the C-terminal domain (C-td) transitions remained unaffected. C-td substitutions led to a more synchronized unfolding/refolding process of the N-td and C-td, and the overall transitions shifted to lower GuHCl concentrations. These results were consistent with a sequential unfolding/refolding model of H[gamma]D-Crys, in which the N-td unfolds first and refolds last. The Greek key pairs had larger contributions to both thermal stability and thermodynamic stability than the non-Greek-key pairs. Aromatic-aromatic interaction energy was estimated by double mutant cycles as 1.5-2.0 kcal/mol. To distinguish the effects in unfolding and refolding, kinetic experiments were also performed. In kinetic unfolding experiments, N-td substitutions accelerated the early phase of unfolding, while C-td substitutions accelerated the late phase. For refolding, only substitutions of the second Greek key pair of each crystallin domain slowed refolding: N-td substitutions Y45A and Y5OA affected the late phase while Y133A and Y138A affected the early phase of the overall refolding reaction. The second Greek key may serve as a nucleation site during the folding of the double-Greek-key crystallin domain. The aggregation pathway that competes with productive refolding in vitro, as well as the suppression of aggregation by chaperone H[alpha]B-Crys were also investigated for mutant H[gamma]D-Crys variants with replacements of the Greek key paired residues. The WT and the mutant H[gamma]D-Crys behaved very similarly, in term of aggregation kinetics and final aggregation level, indicating that these aromatic pairs played minimal role in the aggregation process. The efficiencies of aggregation suppression by H[alpha]B-Crys, as well as the H[alpha]B-bound conformations characterized by the fluorescence of H[gamma]B-H[gamma]D complexes were also very similar for the WT and mutant HyDCrys, arguing against a critical role of these aromatic pairs in the chaperone recognition process. Together with the earlier results, it can also be concluded that stability and refolding kinetics of H[gamma]D-Crys were not critical determinants for its refolding-induced aggregation as well as HaBCrys recognition. Both of these processes may rely on features of the folding intermediate in a very early stage of Greek key refolding. The tryptophan fluorescence of the H[alpha]B-H[gamma]D complexes with WT or mutant H[gamma]D-Crys resembled a partially unfolded state of HyD-Crys, consistent with the tryptophans being part of the contact sites with the chaperone H[alpha]B-Crys.