Photoactive Yellow Protein from the Halophilic Bacterium Salinibacter ruber
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- 作者：Samy Memmi ; John Kyndt ; Terry Meyer ; Bart Devreese ; Michael Cusanovich ; Jozef Van Beeumen
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：February 19, 2008
- 年：2008
- 卷：47
- 期：7
- 页码：2014 - 2024
- 全文大小：277K
- 年卷期：v.47,no.7(February 19, 2008)
- ISSN：1520-4995
文摘
A gene for photoactive yellow protein (PYP) was identified from the genome sequence of theextremely halophilic aerobic bacterium Salinibacter ruber (Sr). The sequence is distantly related to theprototypic PYP from Halorhodospira halophila (Hh) (37% identity) and contains most of the amino acidresidues identified as necessary for function. However, the Sr pyp gene is not flanked by its two biosyntheticgenes as in other species. To determine as to whether the Sr pyp gene encodes a functional protein, wecloned and expressed it in Escherichia coli, along with the genes for chromophore biosynthesis fromRhodobacter capsulatus. The Sr PYP has a 31-residue N-terminal extension as compared to other PYPsthat appears to be important for dimerization; however, truncation of these extra residues did not changethe spectral and photokinetic properties. Sr PYP has an absorption maximum at 431 nm, which is atshorter wavelengths than the prototypical Hh PYP (at 446 nm). It is also photoactive, being reversiblybleached by either blue or white light. The kinetics of dark recovery is slower than any of the PYPsreported to date (4.27 × 10-4 s-1 at pH 7.5). Sr PYP appears to have a normal photocycle with the I1 andI2 intermediates. The presence of the I2' intermediate is also inferred on the basis of the effects of temperatureand alchohol on recovery. Sr PYP has an intermediate spectral form in equilibrium with the 431 nmform, similar to R. capsulatus PYP and the Y42F mutant of Hh PYP. Increasing ionic strength stabilizesthe 431 nm form at the expense of the intermediate spectral form, and the kinetics of recovery is accelerated6.4-fold between 0 and 3.5 M salt. This is observed with ions from both the chaotropic and the kosmotropicseries. Ionic strength also stabilizes PYP against thermal denaturation, as the melting temperature isincreased from 74 
C in buffer alone to 92 C in 2 M KCl. Sr accumulates KCl in the cytoplasm, likeHalobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP isan example of a halophilic protein that requires KCl to electrostatically screen the excess negative chargeand stabilize the tertiary structure.
C in buffer alone to 92 C in 2 M KCl. Sr accumulates KCl in the cytoplasm, likeHalobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP isan example of a halophilic protein that requires KCl to electrostatically screen the excess negative chargeand stabilize the tertiary structure.