Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 A resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP.
Photoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium. Its chromophore (p-coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50. Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state. The structure of E46Q mutant protein was determined at 1.2 A resolution, revealing that the phenolic O atom of p-coumaric acid is hydrogen bonded to NH(2) of Gln46 in E46Q with a longer distance (2.86 +/- 0.02 A) than its distance (2.51 A) to Glu46 OH in the wild type. This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type. Several characteristic features of E46Q such as an alkali shift in the pK(a) and the rapid photocycle can be explained by this weakened hydrogen bond. Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p-coumaric acid owing to the weakening of this hydrogen bond.
Sugishima
Masakazu
M
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka, Osaka 560-0043, Japan.
Tanimoto
Norihiko
N
Soda
Koji
K
Hamada
Norio
N
Tokunaga
Fumio
F
Fukuyama
Keiichi
K
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D003373:Coumaric Acids / Q000737:chemistry; D018360:Crystallography, X-Ray; D020587:Ectothiorhodospira / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry*; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics*; D011422:Propionates; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D013696:Temperature; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; D011422:Propionates; C050162:photoactive yellow protein, Bacteria; D018698:Glutamic Acid; C495469:p-coumaric acid