The eye lens crystallins, which maintain the transparency of the eye lens by providing a well-defined gradient of refractive index, are a scientifically important and medically relevant group of proteins. In contrast to most other proteins, which are constantly subject to degradation and recycling, crystallins have very low turnover and must remain intact for a lifetime. This is even more remarkable considering their extremely high concentration in the lens. Cataract, a major cause of blindness worldwide, results when the structural crystallins aggregate or phase-separate, rendering the lens opaque. Over time, protein degradation occurs when the crystallins become chemically modified, often by deamidation or truncation when damaged by UV light, or by glycation in the case of diabetes. Furthermore, several known point mutations cause hereditary juvenile-onset cataracts. Because of the medical and biophysical relevance of crystallins, there is a need for detailed structural information about both the large complexes formed in the native state and in the cataract- related aggregates. Molecular-level characterization of crystallin aggregation at the level of detail required to guide the design of new therapeutic strategies requires the development of instrumentation and methodology. The objective of this project is to clarify the molecular basis of the crystallin aggregation that leads to cataract formation. The major types of crystallins can be categorized as either structural (b/g) or solubilizing (a). The specific molecular target is gS-crystallin, a major structural component of the eye lens, and its interactions with the ?-crystallin chaperones. New NMR methodology will be developed to investigate the structural factors related to gS-crystallin stability and solubility, primarily in the solid state. Differential isotope labeling of peptide binders and variant crystallins can be used to identify crystallin residues involved in altered intermolecular interactions and provide preliminary structural information. We have designed and built a novel high-field 1H,13C,2H,15N solid-state NMR probe to perform 2H-detected experiments not possible with previously available probes. Building on this success, new experiments that make use of this unique instrumentation will be developed to investigate crystallin aggregates and other solid but highly mobile samples. We will continue to utilize recent advances in solid-state NMR to investigate molecular structure and dynamics in wild-type gS- crystallin at high concentration, aggregates of variants associated with congenital cataracts in humans, as well as aggregates formed by UV irradiation and binding of metal cations. The G18V variant serves as a starting point for our investigations into structure/function relationships in the healthy and cataract states of eye lens proteins; however, in the later stages of the project, the focus of the work will shift to models for age-related cataract, which affects many more patients. Elucidation of these structures will improve our understanding of how cataract formation and guide the development of novel strategies for their prevention and treatment.

Public Health Relevance

The objective of this proposal is to determine how the structural eye lens protein ?S- crystallin aggregates, leading to cataract formation. We will identify aggregation-prone protein variants, characterize the types of aggregates formed, and develop solid-state NMR instrumentation and techniques to determine the molecular structures of both the healthy and cataract states of gS-crystallin. Elucidation of these structures will improve our understanding of cataract formation, potentially leading to novel strategies for their prevention and treatment.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY021514-06
Application #
9662675
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Araj, Houmam H
Project Start
2011-09-01
Project End
2023-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
6
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92617
Martin, Rachel W; Kelly, John E; Kelz, Jessica I (2018) Advances in instrumentation and methodology for solid-state NMR of biological assemblies. J Struct Biol :
Khago, Domarin; Bierma, Jan C; Roskamp, Kyle W et al. (2018) Protein refractive index increment is determined by conformation as well as composition. J Phys Condens Matter 30:435101
Roskamp, Kyle W; Montelongo, David M; Anorma, Chelsea D et al. (2017) Multiple Aggregation Pathways in Human ?S-Crystallin and Its Aggregation-Prone G18V Variant. Invest Ophthalmol Vis Sci 58:2397-2405
Collier, Kelsey A; Sengupta, Suvrajit; Espinosa, Catalina A et al. (2017) Design and construction of a quadruple-resonance MAS NMR probe for investigation of extensively deuterated biomolecules. J Magn Reson 285:8-17
Huang, Kuo-Ying; Kingsley, Carolyn N; Sheil, Ryan et al. (2016) Stability of Protein-Specific Hydration Shell on Crowding. J Am Chem Soc 138:5392-402
Khago, Domarin; Wong, Eric K; Kingsley, Carolyn N et al. (2016) Increased hydrophobic surface exposure in the cataract-related G18V variant of human ?S-crystallin. Biochim Biophys Acta 1860:325-32
Kingsley, Carolyn N; Bierma, Jan C; Pham, Vyvy et al. (2014) ?S-crystallin proteins from the Antarctic nototheniid toothfish: a model system for investigating differential resistance to chemical and thermal denaturation. J Phys Chem B 118:13544-53
Kingsley, Carolyn N; Brubaker, William D; Markovic, Stefan et al. (2013) Preferential and specific binding of human ?B-crystallin to a cataract-related variant of ?S-crystallin. Structure 21:2221-7
Jiang, Jun; Golchert, Kory J; Kingsley, Carolyn N et al. (2013) Exploring the aggregation propensity of ?S-crystallin protein variants using two-dimensional spectroscopic tools. J Phys Chem B 117:14294-301
Brubaker, William D; Martin, Rachel W (2012) ¹H, ¹³C, and ¹?N assignments of wild-type human ?S-crystallin and its cataract-related variant ?S-G18V. Biomol NMR Assign 6:63-7

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