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1.  The C-terminal cysteine annulus participates in auto-chaperone function for Salmonella phage P22 tailspike folding and assembly 
Bacteriophage  2012;2(1):36-49.
Elongated trimeric adhesins are a distinct class of proteins employed by phages and viruses to recognize and bind to their host cells, and by bacteria to bind to their target cells and tissues. The tailspikes of E. coli phage K1F and Bacillus phage Ø29 exhibit auto-chaperone activity in their trimeric C-terminal domains. The P22 tailspike is structurally homologous to those adhesins. Though there are no disulfide bonds or reactive cysteines in the native P22 tailspikes, a set of C-terminal cysteines are very reactive in partially folded intermediates, implying an unusual local conformation in the domain. This is likely to be involved in the auto-chaperone function. We examined the unusual reactivity of C-terminal tailspike cysteines during folding and assembly as a potential reporter of auto-chaperone function. Reaction with IAA blocked productive refolding in vitro, but not off-pathway aggregation. Two-dimensional PAGE revealed that the predominant intermediate exhibiting reactive cysteine side chains was a partially folded monomer. Treatment with reducing reagent promoted native trimer formation from these species, consistent with transient disulfide bonds in the auto-chaperone domain. Limited enzymatic digestion and mass spectrometry of folding and assembly intermediates indicated that the C-terminal domain was compact in the protrimer species. These results indicate that the C-terminal domain of the P22 tailspike folds itself and associates prior to formation of the protrimer intermediate, and not after, as previously proposed. The C-terminal cysteines and triple β-helix domains apparently provide the staging for the correct auto-chaperone domain formation, needed for alignment of P22 tailspike native trimer.
PMCID: PMC3357383  PMID: 22666655
auto-chaperone; cysteines; folding intermediates; tailspike; transient disulfide bond
2.  Solvent accessibility of βB2-crystallin and local structural changes due to deamidation at the dimer interface 
Experimental eye research  2010;91(3):336-346.
In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the β/γ-crystallin family share common β-sheet rich domains and hydrophobic regions at the monomer-monomer or domain-domain interfaces. Disruption of these interfaces, due to post-translational modifications, such as deamidation, decreases the stability of the crystallins. Previous experiments have failed to define the structural changes associated with this decreased stability.
Using hydrogen/deuterium exchange with mass spectrometry (HDMS), deamidation-induced local structural changes in βB2-crystallin were identified. Deamidation was mimicked by replacing glutamines with glutamic acids at homologous residues 70 and 162 in the monomer-monomer interface of the βB2-crystallin dimer. The exchange-in of deuterium was determined from 15 sec to 24 h and the global and local changes in solvent accessibility were measured.
In the wild type βB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of βB2-crystallin in solution. This is consistent with a tightly packed domain structure observed in the crystal structure. Deuterium levels were initially greater in N-terminal domain (N-td) peptides than in homologous peptides in the C-terminal domain (C-td). The more rapid incorporation suggests a greater solvent accessibility of the N-td.
In the βB2-crystallin crystal structure, interface Gln are oriented towards their opposite domain. When deamidation was mimicked at Gln70 in the N-td, deuterium levels increased at the interface peptide in the C-td. A similar effect in the N-td was not observed when deamidation was mimicked at the homologous residue, Gln162, in the C-td. This difference in the mutants can be explained by deamidation at Gln70 disrupting the more compact C-td and increasing the solvent accessibility in the C-td interface peptides.
When deamidation was mimicked at both interface Gln, deuterium incorporation increased in the C-td, similar to deamidation at Gln70 alone. In addition, deuterium incorporation was decreased in the N-td in an outside loop peptide adjacent to the mutation site. This decreased accessibility may be due to newly exposed charge groups facilitating ionic interactions or to peptides becoming more buried when other regions became more exposed.
The highly sensitive HDMS methods used here detected local structural changes in solution that had not been previously identified and provide a mechanism for the associated decrease in stability due to deamidation. Changes in accessibility due to deamidation at the interface led to structural perturbations elsewhere in the protein. The cumulative effects of multiple deamidation sites perturbing the structure both locally and distant from the site of deamidation may contribute to aggregation and precipitation during aging and cataractogenesis in the lens.
PMCID: PMC2926248  PMID: 20639133
lens; β-crystallins; deamidation; hydrogen/deuterium exchange with mass spectrometry; 3D structure
3.  Deamidation alters interactions of β-crystallins in hetero-oligomers 
Molecular Vision  2009;15:241-249.
Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein–protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the βA3-crystallin (βA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of βA3 with other β-crystallin subunits.
Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between βA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type βA3 or the doubly deamidated mutant βA3 Q85E/Q180E (DM βA3) were mixed with either βB1- or βB2-crystallin (βB1 or βB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE.
Molar masses of the hetero-oligomer complexes indicated βA3 formed a polydispersed hetero-tetramer with βB1 and a mondispersed hetero-dimer with βB2. Deamidation at the interface in the βA3 dimer decreased formation of the hetero-oligomer with βB1 and further decreased formation of the hetero-dimer with βB2. During thermal-induced denaturation of the deamidated βA3 dimer, βB1 but not βB2 was able to prevent precipitation of βA3.
Deamidation decreased formation of hetero-oligomers between β-crystallin subunits. An excess accumulation of deamidated β-crystallins in vivo may disrupt normal protein–protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble β-crystallins during aging and cataracts.
PMCID: PMC2633459  PMID: 19190732
4.  Deamidation alters the structure and decreases the stability of human lens βA3-crystallin 
Biochemistry  2007;46(30):8861-8871.
According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging.
The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the βA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far UV and near UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to wild type βA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat-denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the βA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions.
PMCID: PMC2597435  PMID: 17616172

Results 1-4 (4)