UC Irvine

βγ-crystallins are a diverse family of proteins characterized by a conserved Greek-key fold and found across a wide range of organisms. Although vertebrate βγ-crystallins are best known as structural proteins of the eye lens, members of this family are thought to have evolved from ancestral calcium-binding proteins. The Ciona intestinalis βγ-crystallin (Ciβγ) is particularly informative for understanding the evolution of this protein family because it retains calcium-binding properties while maintaining similar structural features to newer vertebrate lens βγ-crystallins. Understanding how calcium binding influences the structure and dynamics of Ci-βγ therefore provides insight into molecular properties that may have accompanied the evolution of the βγ-crystallin family. This dissertation uses nuclear magnetic resonance (NMR) spectroscopy to characterize the structural, calcium-binding, and dynamic properties of Ci-βγ. Backbone and side-chain resonance assignments were established for the protein, providing the foundation for residue-specific characterization of its molecular properties. Calcium-dependent changes were investigated using 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy and chemical shift perturbation analysis, allowing the effects of calcium binding to be monitored throughout the protein. These studies demonstrate that calcium binding produces localized changes in the chemical environment of residues surrounding the calcium-binding sites while also influencing regions outside the immediate coordination sphere. 15N relaxation measurements were used to characterize the backbone dynamics of Ci-βγ in both calcium-bound and calcium-free states. Longitudinal relaxation rates (R1), transverse relaxation rates (R2), and heteronuclear NOE measurements were used to evaluate residue-specific motions on picosecond-to-nanosecond timescales. ModelFree analysis was used to estimate generalized order parameters and identify regions exhibiting differences in backbone flexibility. Comparison of the apo and calcium-bound states reveals how calcium binding influences the dynamic behavior of the protein. Together, these NMR studies provide a comprehensive residue-specific characterization of the structure, calcium-dependent behavior, and dynamics of Ciona intestinalis βγ-crystallin. The results demonstrate how calcium binding contributes to local structural organization and broader conformational dynamics, providing insight into the molecular characteristics of an evolutionarily relevant βγ-crystallin and the evolution of calcium-binding proteins into the diverse βγ-crystallin family.