title: Assessing the correlation between mutant rhodopsin stability and the severity of retinitis pigmentosa.
abstract: OBJECTIVE
  Following a previous study that demonstrated a correlation between rhodopsin stability and the severity of retinitis pigmentosa (RP), we investigated whether predictions of severity can be improved with a regional analysis of this correlation. The association between changes to the stability of the protein and the relative amount of rhodopsin reaching the plasma membrane was assessed.
  
  
  METHODS
  Crystallography-based estimations of mutant rhodopsin stability were compared with descriptions in the scientific literature of the visual function of mutation carriers to determine the extent of associations between rhodopsin stability and clinical phenotype. To test the findings of this analysis, three residues of a green fluorescent protein (GFP) tagged rhodopsin plasmid were targeted with site-directed random mutagenesis to generate mutant variants with a range of stability changes. These plasmids were transfected into HEK-293 cells, and then flow cytometry was used to measure rhodopsin on the cells' plasma membrane. The GFP signal was used to measure the ratio between this membrane-bound rhodopsin and total cellular rhodopsin. FoldX stability predictions were then compared with the surface staining data and clinical data from the database to characterize the relationship between rhodopsin stability, the severity of RP, and the expression of rhodopsin at the cell surface.
  
  
  RESULTS
  There was a strong linear correlation between the scale of the destabilization of mutant variants and the severity of retinal disease. A correlation was also seen in vitro between stability and the amount of rhodopsin at the plasma membrane. Rhodopsin is drastically reduced on the surface of cells transfected with variants that differ in their inherent stability from the wild-type by more than 2 kcal/mol. Below this threshold, surface levels are closer to those of the wild-type.
  
  
  CONCLUSIONS
  There is a correlation between the stability of rhodopsin mutations and disease severity and levels of membrane-bound rhodopsin. Measuring membrane-bound rhodopsin with flow cytometry could improve prognoses for poorly characterized mutations and could provide a platform for measuring the effectiveness of treatments.
authors: Department of Molecular Ophthalmology, Lions Eye Institute, Perth, Western Australia ; Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia.
authors: Richard
authors: R
authors: McKeone
authors: Centre for Experimental Immunology, Lions Eye Institute, Perth, Western Australia ; Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia.
authors: Matthew
authors: M
authors: Wikstrom
authors: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain ; Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors: Christina
authors: C
authors: Kiel
authors: Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia.
authors: P Elizabeth
authors: PE
authors: Rakoczy
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D055550:Protein Stability; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000503:physiopathology; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics*; D013223:Statistics as Topic; D014162:Transfection; D014792:Visual Acuity
chemical_list: D050505:Mutant Proteins; D049452:Green Fluorescent Proteins; D012243:Rhodopsin