Introduction of a rod pigment aromatic cluster does not improve the structural stability of the human green cone pigment.
In the course of our studies on the structure/function relationship of visual pigments, we have expressed the human green cone pigment in the baculovirus/insect cell expression system. Purification of the human green cone pigment, however, has so far proven to be severely hampered by the low thermal stability of this receptor in a solubilized state. In order to overcome this problem, we tested a variety of chemical compounds that have been described to improve protein stability in various applications. The presence of glycerol, sucrose, trehalose and lipids during extraction improved the thermal stability of the recombinant green cone pigment up to twofold. We also analyzed the effect of mutation of residues Met208, Cys212 and Cys273 into Phe in all combinations. These mutants were designed in an attempt to increase the thermal stability by replacing weakly interacting side chains in the green pigment with their counterparts in rhodopsin with strong aromatic stacking interaction. All mutants produced wild-type levels of functional pigment, but none showed an increase in thermal stability.
Giesbers
Maikel E
ME
Department of Biochemistry, UMC-Nijmegen and Nijmegen Center for Molecular Life Sciences, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands.
Bosman
Giel J C G M
GJ
Bovee-Geurts
Petra H M
PH
DeGrip
Willem J
WJ
D000818:Animals; D001483:Base Sequence; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry*; D012168:Retinal Pigments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry*; D016415:Sequence Alignment; D013395:Sucrose / Q000737:chemistry; D014199:Trehalose / Q000737:chemistry
D008055:Lipids; D011994:Recombinant Proteins; D012168:Retinal Pigments; D013395:Sucrose; D014199:Trehalose; D005990:Glycerol