Aggregation of beta A3-crystallin is independent of the specific sequence of the domain connecting peptide.

Hope, J.; Chen, Hao-Chia; Hejtmancik, J. F.

doi:10.1016/s0021-9258(17)31941-5

Cited by 25 publications

(10 citation statements)

References 32 publications

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“…This would be manifest as a compacting of the protein structure resulting in a lower apparent molecular size as estimated by gel filtration. Regardless, we have included the gel filtration data for comparison with our earlier work (26,28). Overall, our ultracentrifugation data clearly support the view that the loss of the N-terminal extension of rβA3 increases its affinity to associate.…”

Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

“…Protein Expression and Purification. Wild-type and modified recombinant murine βA3-crystallins were prepared as previously described ( , ). In rβA3tr, N-terminal residues 1−29 were removed and residue 30 was mutated (W30G).…”

Section: Methodsmentioning

confidence: 99%

“…Also, insertion of the bovine γB-linker into βB2-crystallin, thus, replacing the normal linker, results in monomer formation (27). In contrast, insertion of the γB linker into the murine βA3 does not inhibit dimer formation (28), and bovine γB with βB2-linker inserted is still monomeric (20).…”

mentioning

confidence: 99%

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Monomer−Dimer Equilibrium of Normal and Modified βA3-Crystallins: Experimental Determination and Molecular Modeling

Wingfield

2000

Biochemistry

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and γ-Crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for clarity and refraction. Two regions of the γ-crystallins have been suggested to modulate protein association, namely, the flexible N-terminal extensions and the intramolecular domain interfaces. The oligomeric state of wild-type recombinant murine A3-crystallin (r A3) was compared to that of modified A3-crystallins with either an N-terminal deletion of residues 1 to 29 (r A3tr) or with residues 114 to 123 of the interdomain linker replaced with the analogous linker from murine γB-crystallin (r A3cp). All three proteins exhibited reversible monomerdimer formation. The modifications to the N-terminus and domain linker resulted in tighter dimer formation as compared to wild-type protein as indicated by disassociation constants determined by sedimentation equilibrium: 6.62 × 10 -6 M (r A3), 0.86 × 10 -6 M (r A3cp), and 1.83 × 10 -7 M (r A3tr). Homology modeling of A3-crystallins and solvation energy calculations also predicted tighter binding of the modified crystallins consistent with the centrifugation results. The findings suggest that under physiological conditions A3 crystallin exists in a dynamic equilibrium between monomeric and dimeric protein and that modification, especially to the N-terminal extension, can promote self-association.Crystallins make up over 90% of the water-soluble protein of the mammalian eye lens, where they are critical for lens transparency and refraction. In the vertebrate eye lens, three major classes of ubiquitous crystallins are found: R-, -, and γ-crystallins (1, 2). The -and γ-crystallins have a common polypeptide chain fold, share conserved sequences, and together form a superfamily of γ-crystallins (3). In contrast, the R-crystallins form a separate family of proteins related to the small heat-shock proteins (4). Mutations in γ-crystallin genes can lead to nonspecific aggregation of the γ-crystallins resulting in cataract formation (5-12) as can mutations that alter γD-crystallin association without changing the polypeptide chain fold (13,14).γ-Crystallins comprise two domains connected by an 8-10 amino acid interdomain linker. Each domain has an identical polypeptide chain fold, namely, a -sandwich of two antiparallel -sheets, known as a "Greek key" motif (15-19). The relative position of the two domains in γB-and B2-crystallins differ, having either "closed" or "opened" conformations, respectively (20). The surface location of the interdomain interface in both bovine γΒ and B2 is very similar (19, 21). However, in monomeric γB, intramolecular domains form the interface, whereas in homodimeric B2, the interface consists of residues from the N-terminal domain of one monomer and residues from the C-terminal domain of the second monomer in a switched domain fashion. The physical properties of -sheet residues forming the interdomain interface in A3 crystallin ( A3) are similar to those in γB and B2, especially those residues showi...

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Monomer−Dimer Equilibrium of Normal and Modified βA3-Crystallins: Experimental Determination and Molecular Modeling

Wingfield

2000

Biochemistry

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“…Similarly, truncated forms of βA3-crystallin were found in lens proteins of aging mice (48) and in the soluble protein of young rat (49) lenses. Furthermore, age-dependent cleavage of the C-terminal extension of bovine βB2-crystallin (50) and at the N-terminal extensions of βA3-or βB2-crystallins have been reported (29,30,(51)(52)(53)(54). Terminal extensions are proteolytically processed by thiol proteases (52,53).…”

Section: Discussionmentioning

confidence: 99%

Energetics of Domain−Domain Interactions and Entropy Driven Association of β-Crystallins

2003

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Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.

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β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

2009

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Human age-onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens cD-crystallin protein and its isolated domains. A partially unfolded folding intermediate of cD-crystallin is detected in simulations with its C-terminal domain (C-td) folded and N-terminal domain (N-td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N-td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C-td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non-native surface salt-bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non-native Glu135-Arg142 salt-bridge causes significant destabilization to the folding core of the isolated C-td, which, in turn, induces unfolding of the N-td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of cD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the cD-crystallin surface might lead to unfolding and subsequent aggregation.

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Aggregation of beta A3-crystallin is independent of the specific sequence of the domain connecting peptide.

Cited by 25 publications

References 32 publications

Monomer−Dimer Equilibrium of Normal and Modified βA3-Crystallins: Experimental Determination and Molecular Modeling

Monomer−Dimer Equilibrium of Normal and Modified βA3-Crystallins: Experimental Determination and Molecular Modeling

Energetics of Domain−Domain Interactions and Entropy Driven Association of β-Crystallins

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

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