Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress

Chen, Ya-Huei; Lee, Ming-Ting; Cheng, Yeung Leung; Chou, Wei-Yuan; Yu, Chung-Ming; Lee, Hwei-Jen

doi:10.1016/j.biochi.2010.10.003

Cited by 4 publications

(1 citation statement)

References 32 publications

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“…coli BL21 (DE3) with induction by isopropyl-β- D -thiogalactopyranoside (IPTG). Proteins were purified as previously described [ 8 , 17 ]. The supernatants of crude cell extracts were loaded onto a Q-Sepharose anion exchange column (HiPrep 16/10 Q XL, GE Healthcare) pre-equilibrated in buffer A (50 mM Tris-HCl buffer, pH 7.5) and eluted with a linear gradient of 0 to 0.4 M NaCl in buffer A. Recombinant protein was eluted at approximately 0.15 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly

Huang

Lin

Chou³

et al. 2016

PLoS ONE

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δ-Crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. This protein is structurally assembled as double dimers. Lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. This study found that wild-type protein had both dimers and monomers present in 2–4 M urea whilst only monomers of the K315A mutant were observed under the same conditions, as judged by sedimentation velocity analysis. The assembly of monomeric K315A mutant was reversible in contrast to wild-type protein. Molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. These results suggest the critical role of Lys-315 in stabilization of the diagonal dimer structure. Guanidinium hydrochloride (GdmCl) denatured wild-type or K315A mutant protein did not fold into functional protein. However, the urea dissociated monomers of K315A mutant protein in GdmCl were reversible folding through a multiple steps mechanism as measured by tryptophan and ANS fluorescence. Two partly unfolded intermediates were detected in the pathway. Refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. The formation of aggregates was not prevented by the addition of α-crystallin. These results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs.

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Section: Methodsmentioning

confidence: 99%

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly

Huang

Lin

Chou³

et al. 2016

PLoS ONE

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Identification of compound–protein interactions through the analysis of gene ontology, KEGG enrichment for proteins and molecular fragments of compounds

et al. 2016

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Compound-protein interactions play important roles in every cell via the recognition and regulation of specific functional proteins. The correct identification of compound-protein interactions can lead to a good comprehension of this complicated system and provide useful input for the investigation of various attributes of compounds and proteins. In this study, we attempted to understand this system by extracting properties from both proteins and compounds, in which proteins were represented by gene ontology and KEGG pathway enrichment scores and compounds were represented by molecular fragments. Advanced feature selection methods, including minimum redundancy maximum relevance, incremental feature selection, and the basic machine learning algorithm random forest, were used to analyze these properties and extract core factors for the determination of actual compound-protein interactions. Compound-protein interactions reported in The Binding Databases were used as positive samples. To improve the reliability of the results, the analytic procedure was executed five times using different negative samples. Simultaneously, five optimal prediction methods based on a random forest and yielding maximum MCCs of approximately 77.55 % were constructed and may be useful tools for the prediction of compound-protein interactions. This work provides new clues to understanding the system of compound-protein interactions by analyzing extracted core features. Our results indicate that compound-protein interactions are related to biological processes involving immune, developmental and hormone-associated pathways.

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Real-time heterogeneous protein–protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM)

et al. 2015

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The lens transparency depends on higher concentration of lens proteins and their interactions. α-Crystallin is one of the predominant lens proteins, responsible for proper structural and functional architecture of the lens microenvironment, and any alteration of which results in cataract formation. The R12C, R21L, R49C and R54C are the most significant and prevalent αA-crystallin congenital cataract-causing mutants worldwide. Protein-protein interaction, crucial for lens proper structure and function, was posited to be lost due to point mutation and the elucidation of which could shed light on the molecular basis of cataract. In this conjuncture, we report quartz crystal microbalance (QCM) as a warranted technique for real-time analysis of protein-protein interaction between the N-terminal mutants of αA-crystallin and αB-crystallin. The biophysical characteristics of the mutated proteins were determined by size-exclusion HPLC, far-UV circular dichroism and fluorescence studies. Far-UV circular dichroism spectral analysis displayed slight modifications in β-sheet of R54C mutant. Altered intrinsic tryptophan fluorescence and decreased bis-ANS fluorescence were observed in all the N-terminal mutations revealing the tertiary structural changes and decreased exposure of surface hydrophobicity. An emphatic fall in the chaperone activity was observed in the N-terminal mutants, R12C, R21L and R54C. QCM analysis revealed the occurrence of strong heterogeneous interaction between αA-crystallin and αB-crystallin. Nevertheless, decreased interactions were observed with the N-terminal mutants. In summary, the present study concludes that the loss of interactions between αA-crystallin N-terminal mutants and αB-crystallin signifies quaternary structural alterations due to mutation in the arginine residues.

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Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress

Cited by 4 publications

References 32 publications

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly

Identification of compound–protein interactions through the analysis of gene ontology, KEGG enrichment for proteins and molecular fragments of compounds

Real-time heterogeneous protein–protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM)

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