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 lase r excitation at the 446-nm wavelength maximum, during which the trans- 4-hydroxycinnamic acid chromophore (covalently bound via a thioester t o Cys 69) is isomerized, causing the protein to undergo a conformation al change. We have reconstituted the holoprotein from recombinant apop rotein plus thiophenol thioester-activated chromophore and have also s uccessfully attached a synthetic 3,4-dihydroxycinnamic acid chromophor e and purified the resultant variant, The reconstituted recombinant pr otein has the same spectral and photochemical properties as the native protein. However, the absorption maximum of the protein with the dihy droxy 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 p roteins are too large to measure with the present apparatus, The secon d bleaching phase is only marginally accessible in the variant and has a rate constant (k approximate to 2.3 x 10(4) s(-1)) at least an orde r of magnitude larger than that of the native PYP. In contrast, the ra te constant for recovery of absorbance in the variant (k approximate t o 0.15 s(-1)) is about 40-fold smaller than for native PYP and is inse nsitive to pH (the native protein has a biphasic 16-fold variation in rate constant with pH). We previously observed similar changes in kine tic rate constants for protein denatured by urea or alcohols, which su ggests that the dihydroxy protein is less stable than the native PYP. This was confirmed by measurement of protein unfolding in guanidine hy drochloride. We conclude from these results that the binding site is t oo small to accommodate the dihydroxybenzene ring of the variant chrom ophore without introducing strain into the protein, which is then refl ected in the kinetic properties of the photocycle. (C) 1997 Academic P ress.