Glutamine Deamidation Destabilizes Human γD-Crystallin and Lowers the Kinetic Barrier to Unfolding

Flaugh, Shannon L.; Mills, Ira; King, Jonathan

doi:10.1074/jbc.m603882200

Cited by 113 publications

(177 citation statements)

References 67 publications

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“…Therefore, our simulations reveal that the interdomain interactions contribute to the stability of the N-td of cD-crys, in line with the experimental observation that the mutations weakening interdomain interactions lower both the stability and the folding rate of the N-td, while the C-td remains unaffected. [12][13][14] To obtain a more detailed view of the conformational changes during the unfolding processes, we monitored the secondary structure change as a function of time for the isolated domains, as well as of the full monomer (Supporting Information Fig. S1).…”

Section: Resultsmentioning

confidence: 99%

“…The crucial role of the domain interface interactions in the folding, stability and oligomerization of both b-and c-crystallins is also evident from recent experimental results. [9][10][11][12][13][14] In the crowded lens nucleus, those interactions at the domain-domain surface are likely crucial in preventing incorrect protein-protein association leading to cataract formation.…”

Section: Introductionmentioning

confidence: 99%

“…[9][10][11][12][13][14] The individual domains of cD-crys exhibit differential stability with the C-td being more stable than the N-td. [16][17][18] Equilibrium unfolding/refolding experiments of cD-crys at near-physiologic conditions (at neutral pH and 37 C using Guanidinium Chloride [GdmCl] as denaturant) has suggested sequential folding of its domains with the C-td refolding first.…”

Section: Introductionmentioning

confidence: 99%

“…19 Site-specific mutagenesis experiments demonstrated that the domain interface residues nucleate the N-td folding. [12][13][14] Although both isolated domains could refold efficiently by themselves, the N-td exhibited lower stability than the C-td in isolation. 20 Stability comparisons of the isolated domains with the full monomer revealed that the domain interface contributes a DG H2O of $4.2 kcal/mol to the stability of the full cD-crys monomer.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Although both cooperative folding and independent folding of domains have been observed (11), whether topology determines the cooperativity has not yet been clarified. To shed light on this problem, we here analyze the folding of the example proteins human ␥D-crystallin (17)(18)(19)(20)(21)(22), protein S (23,24), and a tandem array of the R16 and R17 domains of spectrin (25)(26)(27)(28) by using a simple structure-based model and show that the model indeed captures the essential features of the folding of these multidomain proteins.…”

mentioning

confidence: 99%

Cooperativity, connectivity, and folding pathways of multidomain proteins

Itoh

Sasai

2008

Proc. Natl. Acad. Sci. U.S.A.

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Multidomain proteins are ubiquitous in both prokaryotic and eukaryotic proteomes. Study on protein folding, however, has concentrated more on the isolated single domains of proteins, and there have been relatively few systematic studies on the effects of domain-domain interactions on folding. We here discuss this issue by examining human ␥D-crystallin, spore coat protein S, and a tandem array of the R16 and R17 domains of spectrin as example proteins by using a structure-based model of folding. The calculated results consistently explain the experimental data on folding pathways and effects of mutational perturbations, supporting the view that the connectivity of two domains and the distribution of domain-domain interactions in the native conformation are factors to determine kinetic and equilibrium properties of cooperative folding.energy landscape theory ͉ structure-based model ͉ circular permutation O ur understanding of protein folding has been deepened by the combined efforts of experimental, theoretical, and computational studies of the last decade. Energy landscape theory describes folding as the stochastic relaxation process on the free energy surface of conformational change, where the free energy surface is determined by the compromise of conformational entropy of the polymer chain and interaction energies to stabilize the native conformation (1-3). Because proteins can fold when interactions that stabilize the native conformation dominate over the nonnative interactions that may trap the chain into the irrelevant structures, protein folding can be approximately simulated by using the interaction potentials that are derived from the knowledge of the native structure. With such structure-based models, folding of various small proteins has been simulated, and quantitative agreement between simulations and experiments has been reported (3). The agreement has been improved by simulations that further take account of the residue-dependent energetic differences (4), the atomistic packing (5-7), or the hydration structure (8), and such agreement has convinced us that the topology of the native structure is the primary determinant of the equilibrium and kinetic features of folding at least for small proteins (3) although the atomistic details perturb those features or they sometimes change the delicate balance among folding pathways (9, 10).These intensive studies on folding have been predominantly focused on small, single-domain proteins or isolated single domains of larger proteins. More than 70% of eukaryotic proteins, however, are composed of multiple domains, and hence we should ask whether the principles of folding found in single domains of proteins also apply to connected multidomain proteins as well (11). Anticorrelation between the contact order and the folding rate has been observed in multidomain proteins (12), and the structure-based simulations on multidomain proteins such as the ankyrin family (13-15) and CV-N (16) have provided consistent results with experiments. The importance of interactions...

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Cataract as a Protein‐Aggregation Disease

Wang

King

2010

Protein Misfolding Diseases

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Glutamine Deamidation Destabilizes Human γD-Crystallin and Lowers the Kinetic Barrier to Unfolding

Cited by 113 publications

References 67 publications

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

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

Cooperativity, connectivity, and folding pathways of multidomain proteins

Cataract as a Protein‐Aggregation Disease

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