Subunit exchange of lens α‐crystallin: a fluorescence energy transfer study with the fluorescent labeled αA‐crystallin mutant W9F as a probe

The chaperone activity of native ␣-crystallins toward ␤ LOW -and various ␥-crystallins at the onset of their denaturation, 60 and 66°C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human ␥S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of ␣-crystallins at temperatures above 60°C. Further increase is observed at 66°C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These ␣-crystallin temperature-induced modifications were found necessary to allow for the association with ␤ LOW -and ␥-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)-and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified ␣-crystallins retained their ability to exchange subunits and that, at 37°C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66°C. Association with ␤ LOW -(60°C) or various ␥-crystallins (66°C) resulted at 37°C in decreased subunit exchange in proportion to bound ligands. Therefore, ␤ LOW -and ␥-crystallins were compared for their capacity to associate with ␣-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual ␥-crystallin family members. The strongest effect was observed for ␥S, followed by h␥Srec, ␥E, ␥A-F, ␥D, ␥B. Moreover, fluorescence properties of ␣-crystallins in the presence of bound ␤ LOW -and ␥-crystallins indicated that the formation of ␤ LOW /␣-or ␥/␣-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of ␣-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated ␣-crystallin structure.␣-Crystallins and small heat shock proteins have in common a conserved C-terminal domain of about 115 residues, the ␣-crystallin domain, the structure of which is organized around an eight-stranded ␤-sandwich (1, 2). The small heat shock proteins usually associate into high molecular weight monodisperse or polydisperse oligomers, able to protect against stress through the binding of a variety of partially unfolded substrates. ␣-Crystallin was demonstrated by Horwitz in 1992 (3) to also exhibit chaperone properties in vitro. It is able to bind ␤-and ␥-crystallins at the onset of their thermal denaturation, thus preventing further denaturation and aggregation, yet not refolding. Following this pioneering work, the chaperone-like activity of ␣-crystallin has been the subject of numerous studies and functional models have been suggested (4 -30). Essentially, hydrophobic patches on the surface, either present in the native state or revealed after structural modifications, would associate w...

Section: ␣-Crystallin Chaperone Activity and Subunit Exchangementioning

confidence: 59%

mentioning

confidence: 99%

Subunit Exchange Demonstrates a Differential Chaperone Activity of Calf α-Crystallin toward βLOW- and Individual γ-Crystallins

Putilina¹,

Skouri‐Panet²,

Prat³

et al. 2003

“…Although the exact role of this dynamic property is not clear, it appears to be of some functional importance, since ␣B-crystallin and Hsp25/27 form heteromultimers with one another in vivo (47)(48)(49)(50). We therefore studied the dynamic property of subunit exchange of the wild type and the deletion mutants.…”

Section: Fig 3 Sedimentation Of the Wild Type ␣-Crystallins And Thementioning

confidence: 99%

Role of the Conserved SRLFDQFFG Region of α-Crystallin, a Small Heat Shock Protein

Pasta

Raman

Ramakrishna

et al. 2003

Small heat shock proteins (sHsps) are necessary for several cellular functions and in stress tolerance. Most sHsps are oligomers; intersubunit interactions leading to changes in oligomeric structure and exposure of specific regions may modulate their functioning. Many sHsps, including ␣A-and ␣B-crystallin, contain a well conserved SRLFDQFFG sequence motif in the N-terminal region. Sequence-based prediction shows that it exhibits helical propensity with amphipathic character, suggesting that it plays a critical role in the structure and function of ␣-crystallins. In order to investigate the role of this motif in the structure and function of sHsps, we have made constructs deleting this sequence from ␣A-and ␣B-crystallin, overexpressed, purified, and studied these engineered proteins. Circular dichroism spectroscopic studies show changes in tertiary and secondary structure on deletion of the sequence. Glycerol density gradient centrifugation and dynamic light scattering studies show that the multimeric size of the mutant proteins is significantly reduced, indicating a role for this motif in higher order organization of the subunits. Both deletion mutants exhibit similar oligomeric size and increased chaperone-like activity. Urea-induced denaturation study shows that the SRLFDQFFG sequence contributes significantly to the structural stability. Fluorescence resonance energy transfer studies show that the rate of exchange of the subunits in the ␣Adel-crystallin oligomer is higher compared with that in the ␣A-crystallin oligomer, suggesting that this region contributes to the oligomer dynamics in addition to the higher order assembly and structural stability. Thus, our study shows that the SRLFDQFFG sequence is one of the critical motifs in structure-function regulation of ␣A-and ␣B-crystallin.␣-Crystallin, a multimeric protein composed of two types of subunits, ␣A-and ␣B-crystallin, is abundantly present in the eye lens (1). Both ␣A-and ␣B-crystallin have subunit molecular masses of ϳ20 kDa each, share high sequence homology (2), can form homomultimers (3, 4), and are known to be present in other nonlenticular tissues as well (5, 6). Ingolia and Craig (7), while comparing the sequences of four small heat shock proteins from Drosophila, discovered the remarkable similarity between ␣B-crystallin and small heat shock proteins.Prompted by this sequence similarity, Klemenz et al. (8) investigated the expression of ␣-crystallin under heat shock and concluded that indeed ␣-crystallin is a heat shock protein. Horwitz (9) subsequently showed that ␣-crystallin prevents aggregation of other proteins like other heat shock proteins.␣B-crystallin and not ␣A-crystallin is stress-inducible. Its levels increase under stress such as heat shock, ischemia, oxidation, and infection and in various disease conditions (10 -12). ␣A-and ␣B-crystallin, either in their hetero-or homo-oligomeric states, exhibit molecular chaperone-like properties in preventing protein aggregation (4,9,13,14). ␣-Crystallin binds to partially unfolded, aggregatio...

“…They are present in solution as 600 -800-kDa homo-oligomers. We have also reported some of their biochemical and biophysical properties (17,22,23). Protein concentrations were determined by absorption based on aromatic amino acid composition (24).…”

Section: Preparation Of Recombinant ␣-Crystallin Samplesmentioning

confidence: 99%

“…We have reported differences in hydrophobicity, chaperonelike activity, thermal stability, and rate of subunit exchange between ␣A-and ␣B-crystallins (17,22,23). The thermal stability study indicated that ␣B-crystallin is more susceptible to aggregation than ␣A-crystallin and that addition of ␣A-to ␣B-crystallin reduces aggregation (22).…”

Section: Dissociation Study By Fplc and Lightmentioning

confidence: 99%

Thermodynamic Stability of Human Lens Recombinant αA- and αB-crystallins

Sun

Akhtar

Liang

1999

Self Cite

Lens ␣-crystallin is a 600 -800-kDa heterogeneous oligomer protein consisting of two subunits, ␣A and ␣B. The homogeneous oligomers (␣A-and ␣B-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both ␣A-and ␣B-crystallins. In terms of standard free energy (⌬G NU H2O ), ␣A-crystallin is slightly more stable than ␣B-crystallin. The significance of the intermediates may be related to the functioning of ␣-crystallins as chaperonelike molecules.Human lens proteins become progressively less soluble with age and cataract formation. The accumulation of high molecular mass aggregated and insoluble proteins was thought to be the main cause of lens opacity (1, 2). The major crystallin in high molecular mass and insoluble proteins, ␣-crystallin, has been thought to play a major role in the maintenance of lens transparency (3), as manifested by the recent finding that ␣-crystallin acts like a chaperone in in vitro experiments (4). However, the mechanisms of high molecular mass aggregation and insolubilization have not been well established. A reasonable assumption is that these events are related to proteins being in an unstable or not fully folded state, and thus tending to aggregate when their concentrations are high. A study on thermodynamic and kinetic stability may provide a reliable clue.An earlier report indicated that the native ␣-crystallin is thermally very stable (5). It does not denature even at 100°C, but undergoes a thermal transition at 60 -65°C. Later, it was reported that ␣-crystallin becomes partially unfolded at this temperature (6). The refolding is irreversible after exposure to the high temperature (6, 7). On the other hand, the reversibility of denaturant unfolding of ␣-crystallin has been reported (8, 9). The thermodynamic and kinetic stability, however, has not been studied in detail, presumably because of the heterogeneity and oligomeric nature of ␣-crystallin. In contrast, the monomeric ␥-crystallin has been extensively studied (10 -13). Recently, we reported a thermodynamic and kinetic study on ␥F-crystallin (14), which is the most stable ␥-crystallin gene product (15,16).To obtain pure homogeneous ␣-crystallin, we have cloned human lens ␣A-and ␣B-crystallins (17), which are good models for studying the unfolding and refolding properties of ␣-crystallin. In addition to the unfolding intermediate, we also need to consider whether ␣-crystallin has a dissociation intermediate (18). In this work, we used the chemica...