May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Characterisation of the Structural Properties of Normal and Mutant ßigh3
Author Affiliations & Notes
  • T.A. Jowitt
    Wellcome Trust Centre for Cell Matrix Research, Research Group in Eye and Vision Science, University of Manchester, United Kingdom
  • P.N. Bishop
    Wellcome Trust Centre for Cell Matrix Research, Research Group in Eye and Vision Science, University of Manchester, United Kingdom
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 420. doi:
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      T.A. Jowitt, P.N. Bishop; Characterisation of the Structural Properties of Normal and Mutant ßigh3 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):420.

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

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Abstract

Abstract: : Purpose: To determine whether there are structural differences between wild type (WT) human ßigh3 and the 5q31-linked mutations R555W and R555Q, which are associated with corneal dystrophies. Methods: Full length WT, R555Q and R555W were expressed as secreted proteins by 293 EBNA cells. A poly-histidine tag was incorporated into all of the constructs and the recombinant proteins were purified by nickel affinity chromatography, with and without guanidine hydrochloride, and by size exclusion chromatography (SEC). Purified samples were analysed by CD spectroscopy and calibrated SEC to investigate protein structure, and by rotary shadowing electron microscopy. Carbohydrate analysis was performed using Emerald Q-300 glycoprotein stain. Results: Analysis of CD spectra revealed a similar secondary structure between WT and mutant proteins with evidence of α-helices and ß-sheet. The CD spectra were the same after treatment with 6M GuHCl, which demonstrates that these structures could be recovered. WT protein was also shown to be stable in temperatures up to 98°C and there was no evidence of any glycosylation on any of the constructs. Size exclusion chromatography revealed that the WT ßigh3 was not significantly elongated and has a hydrodynamic radius of approximately 4 nm. Electron microscopy showed spherical monomers but there was no evidence of fibrillar structures as previously reported. Conclusions: CD spectroscopy revealed that there was no significant alteration in secondary structure between the mutants and wild type proteins. Furthermore ßigh3 is extremely stable and could not be melted at high temperatures.

Keywords: protein structure/function • extracellular matrix • cornea: basic science 
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