Structures of Differently Aggregated and Precipitated Forms of &amp;#947;B Crystallin: An FTIR Spectroscopic and EM Study

Fatima, Uzma; Sharma, Swati; Guptasarma, Purnananda

doi:10.2174/092986610791760414

Cited by 10 publications

(12 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fibers have mean diameters of approximately ∼6 nm, similar to those observed in TEM images of acid-induced γD-crystallin amyloid fibers (21), but have a relatively wide distributions of diameters. These results are in striking contrast to previous studies on UV-induced aggregates of mammalian γ-crystallins, which did not show the presence of fibers in TEM images despite FTIR evidence for extended vibrational coupling in β-sheets (16). …”

Section: Resultscontrasting

confidence: 99%

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

et al. 2013

View full text Add to dashboard Cite

γD-crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of γD-crystallin. The native β-sandwich domains are not retained upon precipitation by either mechanism. The similarity between the amyloid forming pathway when induced by either UV-B radiation or low pH suggests that it is the propensity for the C-terminal β-sandwich domain to form amyloid β-sheets that determines the misfolding pathway independent of the mechanism of denaturation.

show abstract

Section: Resultscontrasting

confidence: 99%

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

et al. 2013

View full text Add to dashboard Cite

show abstract

“…In fact, amyloid-like infrared spectra of g-crystallins, without the presence of fiber morphology in TEM images, has been observed in vitro. 6 In addition, the presence of intermolecular cross-links in thermally induced gD-crystallin aggregates mirrors the crosslinking observed in heavily damaged, aged lenses. 2,9,75,76 Although the nature of our in vitro cross-links remains to be determined, our results suggest that the presence of intermolecular crosslinks may influence morphologies and molecular structures of lens crystallin aggregates.…”

Section: Discussionmentioning

confidence: 83%

“…The fact that such spectral features are observed for aggregates that form sheets instead of fibers indicates that amyloid‐like aggregation may occur even where fibers are not observed. In fact, amyloid‐like infrared spectra of γ‐crystallins, without the presence of fiber morphology in TEM images, has been observed in vitro . In addition, the presence of intermolecular cross‐links in thermally induced γD‐crystallin aggregates mirrors the cross‐linking observed in heavily damaged, aged lenses .…”

Section: Discussionmentioning

confidence: 93%

“…Cataracts are a common protein aggregation disease of the eye lens, resulting from the formation of insoluble deposits of lens crystallin proteins that blur vision . Aggregation may result from mutations as well as accumulated damage from sources such as UV radiation, oxidative stress, and a variety of post‐translational modifications that induce precipitation or misfolding of lens crystallins. Although the causes of damage are well known, the resulting structures of aggregated lens crystallin proteins are poorly understood.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An alternative structural isoform in amyloid‐like aggregates formed from thermally denatured human γD‐crystallin

2014

View full text Add to dashboard Cite

The eye lens protein cD-crystallin contributes to cataract formation in the lens. In vitro experiments show that cD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the b-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended b-sheets, and use isotope dilution experiments to show that each protein contributes approximately one b-strand to each b-sheet in the aggregates. Using segmental 13 C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to b-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking.

show abstract

“…Numerous modes of aggregation have been proposed for the two-domain structure, including amorphous precipitation, domain-swap oligomerization, and amyloid fibril formation (1,26,27). In vitro, fibrillar deposits of γ-crystallins are created by UV damage, low pH, and chemical and thermal denaturation, although it is unclear to what extent each form (or others) contribute to in vivo cataracts (1,(27)(28)(29). We utilize acid initiated aggregation because it reproducibly generates fibrillar aggregates of γ-crystallins and because low pH conditions encountered during lens development may be relevant to juvenile cataract formation (27).…”

mentioning

confidence: 99%

Two-dimensional IR spectroscopy and segmental ¹³ C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

Moran

Woys

Buchanan

et al. 2012

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

128

307

View full text Add to dashboard Cite

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly 13 C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins. C ataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer's disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6-8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8-10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mech...

show abstract

Structures of Differently Aggregated and Precipitated Forms of γB Crystallin: An FTIR Spectroscopic and EM Study

Cited by 10 publications

References 0 publications

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

An alternative structural isoform in amyloid‐like aggregates formed from thermally denatured human γD‐crystallin

Two-dimensional IR spectroscopy and segmental ¹³ C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

Contact Info

Product

Resources

About

Structures of Differently Aggregated and Precipitated Forms of &#947;B Crystallin: An FTIR Spectroscopic and EM Study

Cited by 10 publications

References 0 publications

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

An alternative structural isoform in amyloid‐like aggregates formed from thermally denatured human γD‐crystallin

Two-dimensional IR spectroscopy and segmental 13 C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

Contact Info

Product

Resources

About

Structures of Differently Aggregated and Precipitated Forms of γB Crystallin: An FTIR Spectroscopic and EM Study

Two-dimensional IR spectroscopy and segmental ¹³ C labeling reveals the domain structure of human γD-crystallin amyloid fibrils