Partial assignments of tunicate βγ-crystallin structure.

Abstract

Tunicate Ciona intestinalis (Ci-βγ-crystallin) is a model to study evolution of vertebrates as it has an evolutionary position between the microbial crystallins and the vertebrate lens proteins. The single-domain βγ-crystallin of the tunicate Ci-βγ-crystallin represents the single-domain ancestor of the vertebrate βγ-crystallin. It has been shown that the tunicate Ci-βγ-crystallin has a high affinity to bind to Ca2+ and that this binding greatly stabilizes the protein both thermally and chemically. However, human γS-crystallin doesn't bind to Ca2+ but it stays stable. NMR spectroscopy is used to study the structure and properties of Ci-βγ-crystallin to better understand the stability and evolutionary progression from ancestral to human lens βγ-crystallins. Residual dipolar couplings (RDCs), derived from 15N-1H in-phase/anti-phase (IPAP) heteronuclear single quantum coherence NMR spectra, provide relative orientations among internuclei vectors. These orientations can be used to build the protein backbone structure. RDCs are obtained by weakly aligning proteins in a magnetic field to create an anisotropic condition. The magnetic dipole-dipole interactions no longer average to zero and give measurable dipolar couplings. RDCs are used along with other experimental NMR data for protein structure determination. Xplor-NIH package will be used for protein structure refinement. Xplor-NIH is a popular software package for biomolecular structure determination. The program takes experimental NMR data such as RDCs and other pre-determined theoretical parameters such as covalent geometry (bond lengths and angles) to find the minimum energy structures.