Introduction of a rod pigment aromatic cluster does not improve the structural stability of the human green cone pigment

摘要

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. (C)) 2007 Elsevier Inc. All rights reserved.