Newbold, Richard J and Deery, Evelyne C and Walker, Caroline E and Wilkie, Susan E and Srinivasan, Narayanaswamy and Hunt, David M (2001) The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy. In: Human Molecular Genetics, 10 (1). pp. 47-54.
The_destabilization_of_hu.pdf - Published Version
Restricted to Registered users only
Download (537Kb) | Request a copy
Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline→leucine mutation at position 50 of human GCAP1 results in cone–rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N‐terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Oxford University Press.|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit|
|Date Deposited:||02 Aug 2011 06:14|
|Last Modified:||02 Aug 2011 06:14|
Actions (login required)