Isolation, reconstitution and functional characterisation of the Rhodobacter sphaeroides photoactive yellow protein

FEBS Letters

Hendriks, Johnny¹  Hellingwerf, Klaas¹  Gensch, Thomas¹  Crielaard, Wim¹  Haker, Andrea¹ 

[1] Laboratory for Microbiology, E.C. Slater Institute, BioCentrum, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands

关键词: Photoactive yellow protein;    Rhodobacter sphaeroides;    PYP;    photoactive yellow protein;    E-PYP;    Ectothiorhodospira halophila PYP;    R-PYP;    Rhodobacter sphaeroides PYP;   

DOI  :  10.1016/S0014-5793(00)02242-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We report the isolation, functional reconstitution and photophysical characterisation of Rhodobacter sphaeroides photoactive yellow protein (PYP), of which the gene was recently cloned. Reconstitution of the his-tagged purified apo-protein with 4-hydroxy-cinnamic acid yields the characteristic blue absorbance at 446 nm, but surprisingly also an absorbance peak at 360 nm. This additional peak is not caused by binding of a second chromophore, as confirmed with mass spectroscopy. Moreover, reconstitution with the ‘locked’ analogue 7-hydroxy-coumarin-3-carboxylic acid yields only a single absorbance peak at 441 nm. The 446 nm and 360 nm species are part of a temperature- and pH-dependent equilibrium. Photoactivation of the protein leads to formation of a blue-shifted intermediate as in other PYPs, with a 100-fold increased groundstate recovery rate (k _pB→pG=500 s⁻¹) compared to E-PYP.

【 授权许可】

Unknown