The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin.
Carboxyl group modification with DCCD and NCD-4 was employed to investigate the chemical environment of the side chains of archaeopsin-1 (aO-1) and bacterioopsin (bO). Some differences were observed between aO-1 and bO. Although DCCD or NCD-4 did not modify aO-1 in bleached membrane, they modified bO in bleached membrane and in mixed DMPC/CHAPS/SDS micelles at neutral pH, thereby affecting the opsin shift and the photocycle of the regenerated chromophore. On the contrary, after solubilization with SDS, aO-1 and bO were modified by DCCD and NCD-4, which decreased the chromophore regeneration. In particular, the reaction of aO-1 in SDS with NCD-4 proceeded in a 1:1 ratio at neutral pH. The fluorescence and CD spectra indicated that the modified site was located in the hydrophobic, asymmetrical region. Lysyl-endopeptidase digestion of NCD-4 modified aO-1 produced a fluorescent fragment and amino acid sequence analysis showed that Asp85 or Asp96 in helix C is a probable candidate for the modified residue at present. Kinetic CD measurements revealed that the introduction of N-acylurea at an Asp residue in helix C did not affect the formation of the transient intermediate but inhibited the side chain packing during refolding.
Sugiyama
Y
Y
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, 464-8602, Japan. ysugiya@bio.nagoya-u.ac.jp
Fujii
K
K
Mukohata
Y
Y
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002234:Carbodiimides; D002942:Circular Dichroism; D004024:Dicyclohexylcarbodiimide; D005456:Fluorescent Dyes; D006216:Halobacteriaceae / Q000737:chemistry; D019617:Halobacterium salinarum / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D017494:Proton Pumps
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D001436:Bacteriorhodopsins; D002234:Carbodiimides; D004024:Dicyclohexylcarbodiimide; D005456:Fluorescent Dyes; C055476:archaerhodopsin protein, Archaea; D017494:Proton Pumps; C017827:bacterio-opsin; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide