Preparation and properties of a 3,4-dihydroxycinnamic acid chromophore variant of the photoactive yellow protein.
Native photoactive yellow protein (PYP) is reversibly bleached by laser excitation at the 446-nm wavelength maximum, during which the trans-4-hydroxycinnamic acid chromophore (covalently bound via a thioester to Cys 69) is isomerized, causing the protein to undergo a conformational change. We have reconstituted the holoprotein from recombinant apoprotein plus thiophenol thioester-activated chromophore and have also successfully attached a synthetic 3,4-dihydroxycinnamic acid chromophore and purified the resultant variant. The reconstituted recombinant protein has the same spectral and photochemical properties as the native protein. However, the absorption maximum of the protein with the dihydroxy chromophore variant is red-shifted to 458 nm, with an additional shoulder at about 342 nm. Following a laser flash, the rate constants for the first phase of bleaching in both the native and the variant proteins are too large to measure with the present apparatus. The second bleaching phase is only marginally accessible in the variant and has a rate constant (k approximately 2.3 x 10(4) s-1) at least an order of magnitude larger than that of the native PYP. In contrast, the rate constant for recovery of absorbance in the variant (k approximately 0.15 s-1) is about 40-fold smaller than for native PYP and is insensitive to pH (the native protein has a biphasic 16-fold variation in rate constant with pH). We previously observed similar changes in kinetic rate constants for protein denatured by urea or alcohols, which suggests that the dihydroxy protein is less stable than the native PYP. This was confirmed by measurement of protein unfolding in guanidine hydrochloride. We conclude from these results that the binding site is too small to accommodate the dihydroxybenzene ring of the variant chromophore without introducing strain into the protein, which is then reflected in the kinetic properties of the photocycle.
Devanathan
S
S
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Genick
U K
UK
Getzoff
E D
ED
Meyer
T E
TE
Cusanovich
M A
MA
Tollin
G
G
D001059:Apoproteins / Q000737:chemistry; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002109:Caffeic Acids / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010777:Photochemistry; D020283:Photoreceptors, Microbial; D011487:Protein Conformation; D011489:Protein Denaturation; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D001059:Apoproteins; D001426:Bacterial Proteins; D002109:Caffeic Acids; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; C040048:caffeic acid