May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
A High-Resolution NMR Study of Human GammaD Crystallin and Its Cataract-Linked P23T Mutant: Small Conformational Changes in P23T Explain Its Low and Retrograde Solubility
Author Affiliations & Notes
  • A. Pande
    Chemistry, Univ at Albany-SUNY, Albany, New York
  • J. Zhang
    Chemistry, Univ at Albany-SUNY, Albany, New York
  • A. Shekhtman
    Chemistry, Univ at Albany-SUNY, Albany, New York
  • J. Pande
    Chemistry, Univ at Albany-SUNY, Albany, New York
  • Footnotes
    Commercial Relationships  A. Pande, None; J. Zhang, None; A. Shekhtman, None; J. Pande, None.
  • Footnotes
    Support  NIH Grant EY10535 (J.P.) and ADA Career Development Award 1-06-CD-23 (A.S.)
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4084. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      A. Pande, J. Zhang, A. Shekhtman, J. Pande; A High-Resolution NMR Study of Human GammaD Crystallin and Its Cataract-Linked P23T Mutant: Small Conformational Changes in P23T Explain Its Low and Retrograde Solubility. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4084. doi: https://doi.org/.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: : We showed earlier [1] that the P23T mutant of human GammaD crystallin (HGD) is less soluble than the parent protein, and also shows retrograde solubility. However, the two proteins show no significant difference in secondary or tertiary structure. In order to define the small conformational differences between the two proteins that could explain the hydrophobic protein-protein interactions in P23T, we examined them using high-resolution NMR spectroscopy.

Methods: : [U-,15N]-labeled and [U-, 15N,13C]- labeled HGD and its P23T mutant were prepared by overexpressing the proteins in E.coli. NMR experiments were conducted using Bruker 500 MHz spectrometer. Backbone 1H{15N}- HSQC spectra of the 2 proteins were recorded, in order to obtain a qualitative measure of their structural integrity. A standard suite of triple resonance experiments was used to assign backbone amide protons and nitrogens, alpha and beta carbons and carbonyls. In this manner, about 90% of the residues in both HGD and its P23T mutant have been assigned. Chemical-shift perturbations observed in the spectra of P23T, as compared to HGD, were mapped to the x-ray structure of HGD (PDB: 1hk0)) to correlate chemical-shift changes with the structural change.

Results: : As expected, the major change due to the Pro23 to Thr mutation is close to the mutation site involving residues Asp21 and His22. The effect on Asp21 appears to be relayed to Arg79 and Asn49 due to their proximity. Since Arg79 is involved in a network of hydrogen bonds including water molecules, this H-bond network is likely to be altered. Besides these, the only other major changes are in the hydrophobic residues Leu 71, Val 75 and Ile 171. These hydrophobic residues in their altered position/conformation in P23T could potentially create surface-exposed hydrophobic patches in conjunction with other neighboring hydrophobic residues.

Conclusions: : We have assigned chemical shifts of more than 90% of the backbone nuclei of HGD and P23T. The data suggest that a change in the H-bonding network, and generation of potential hydrophobic patches that could lead to the altered protein-protein interactions in P23T, causing its lower and retrograde solubility.1. Pande A, Annunziata O, Asherie N, Ogun O, Benedek GB, Pande J: Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin. Biochemistry 2005,44:2491-2500.

Keywords: cataract • crystallins • protein structure/function 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×