In vitro interactions of the small heat shock protein chaperone human [alpha]B-crystallin with its physiological substrates in the lens [gamma]-crystallins
Author(s)Acosta-Sampson, Ligia I
Massachusetts Institute of Technology. Dept. of Biology.
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The passive chaperone a-crystallin, a small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses, along with the [beta][gamma]-crystallins. It is composed of two subunits (~ 20 kDa) aA- and [alpha]B-crystallin (aA- and [alpha]B-Crys), which form a hetero-oligomeric, polydisperse complex of ~ 800 kDa in the lens. Aggregates isolated from mature-onset cataracts, the major cause of sight loss worldwide, contain damaged and misfolded forms of [beta][gamma]-crystallins, as well as a-crystallins. I have studied the chaperone function of human [alpha]B-crystallin interacting with its physiological human [gamma]-crystallin substrates. Human [gamma]D-crystallin ([gamma]D-Crys) and [gamma]C-crystallin ([gamma]C-Crys) are st[alpha]Ble and long-lived mammalian [gamma]-crystallins localized in the lens nucleus. Human [gamma]S-crystallin ([gamma]S-Crys) is [alpha]Bundant in the lens outer cortex. All three [gamma]-crystallins can refold in vitro to their native state after unfolding in high concentrations of guanidine hydrochloride (GdnHCl). However, in buffer or very low denaturant concentrations (< 1 M GdnHCl) aggregation of refolding [gamma]-crystallin intermediates competes with productive refolding. Diluting unfolded [gamma]C-, [gamma]D-, or [gamma]S-Crys to low GdnHCl concentrations (at 100 [mu]g/ml, 37°C) resulted in the protein population partitioning between productive refolding and aggregation pathways. [gamma]D-, [gamma]C- or [gamma]S-Crys protein was allowed to refold and aggregate in the presence of [alpha]B-Crys homo-oligomers at different mass-based ratios of [gamma]-Crys to [alpha]B-Crys. [gamma]D- and [gamma]C-Crys aggregation was suppressed to similar levels, whereas [gamma]S-Crys aggregation was not suppressed as strongly in assays measuring solution turbidity at 350 nm. SEC chromatograms of the products of suppression reactions showed the presence of a high molecular weight chaperone-substrate complex. This complex was still present 4 days after the suppression reaction was initiated. Experiments were performed with the [alpha]B-Crys chaperone added 2, 6, or 10 s, after dilution of unfolded [gamma]D-Crys out of high concentrations of denaturant. The results from these experiments showed that the partially folded, aggregation-prone species that is recognized by [alpha]B-Crys chaperone is populated within the first 10 s after refolding and aggregation were initiated. This time period coincided with the refolding of the C-terminal domain of [gamma]D-Crys as determined from kinetic refolding experiments in vitro. Human [gamma]D-Crys contains four Trp residues with one residue located in each quadrant of the protein. Intrinsic buried Trp fluorescence is quenched in the native state relative to the unfolded state of the protein due to intra-domain partial resonance energy transfer from the highly fluorescent Trp donors (W42 and W130) to the highly quenched acceptor Trps (W68 and W156). The efficient quenching of Trp68 and Trp156 depends on an unusual conformation of the Trp ring with respect to its backbone amide, as well as the presence of two tightly bound H2O molecules with oppositely oriented dipoles. Thus, intrinsic Trp fluorescence is a sensitive reporter of the protein conformation. Using a no-Trp mutant of [alpha]B-Crys (W9F/W60F), the conformation of the bound [gamma]D-Crys substrate in [gamma]D -- [alpha]B complexes was determined from intrinsic Trp fluorescence emission. The emission spectra for the substrate did not coincide with a native or fully unfolded conformation of the [gamma]D-Crys controls. To further characterize the conformation of each domain of [gamma]D-Crys in the substrate-chaperone complex, double-Trp [gamma]D-Crys mutants, which conserved the Trp pair in the N-terminal (W130F/W156F) or the C-terminal (W42F/W68F) domain, while the counterpart pair was changed to Phe, were used as substrates in aggregation suppression reactions. The fluorescence emission spectra for the double-Trp mutants in complex with Trp-less [alpha]B-Crys were similar and they did not coincide with the spectra for their respective native or unfolded double-Trp [gamma]D-Crys controls. These results indicated that the bound substrate remained in a partially folded state with neither domain native-like. Triple-Trp [gamma]D-mutants that conserved the highly fluorescent Trp residue in the N-terminal or C-terminal domains were also used as substrates in suppression of aggregation reactions with Trp-less [alpha]B-Crys chaperone. The fluorescence emission spectra of triple-Trp substrates in the substrate-chaperone complex indicated that these residues were not solvent exposed. These results suggest that Trp neighboring regions could be interacting directly with the [alpha]B-Crys chaperone. To further elucidate the specific region in the [gamma]-crystallins that interacts with [alpha]B-Crys in suppression assays, experiments were performed using single-domain constructs of [gamma]D-Crys. The isolated N-terminal ([gamma]D-Ntd) and C-terminal domains ([gamma]D-Ctd) of [gamma]D-Crys, expressed in E. coli, can refold to a native state upon dilution out of denaturant to low concentrations of GdnHCl. The C-terminal domain aggregated upon refolding out of high concentrations of denaturant, while the N-terminal did not under the same assay conditions. However, when [gamma]D-Ctd and [gamma]D-Ntd were unfolded and refolded together, [gamma]D-Ctd recruited [gamma]D-Ntd into the aggregate. [alpha]B-Crys suppressed the aggregation of the [gamma]D-Ctd and formed [gamma]D-Ctd -- [alpha]B complexes. Using W9F/W60F [alpha]B-Crys, I have determined, through the fluorescence emission of [gamma]D-Ctd tryptophans, that the [gamma]D-Ctd in the [gamma]D-Ctd --[alpha]B complexes was partially folded. Inhibition experiments in which the [gamma]D-Ntd and [gamma]D-Ctd isolated domains were refolded sequentially or simultaneously showed that [alpha]B-Crys preferentially recognized [gamma]D-Ctd. These in vitro results provide a model for how a-crystallin interacts with aggregation-prone substrates in vivo wherein an aggregation-prone region in the C-terminal domain of [gamma]D-Crys is exposed in the aggregation-prone species and this region is recognized by [alpha]B-Crys. These results also provide support for protein unfolding/protein aggregation models for cataract, with a-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood, but becoming saturated with advancing age.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.In title on title-page "[alpha]" and "[gamma]" appear as lower case Greek letters. Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (p. 160-175).
DepartmentMassachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology