Groningen,Groningen,TheNetherlands

Signal Transduction in the Photoactive Yellow Protein.I. Photon Absorption and the Isomerization of the Chromophore

Gerrit Groenhof,1Marc F.Lensink,2Herman J.C.Berendsen,1Jaap G.Snijders,3and Alan E.Mark1*

1Department of Biophysical Chemistry,Groningen Biomolecular Sciences and Biotechnology Institute,Rijksuniversiteit Groningen,Groningen,The Netherlands

2Department of Biochemistry,the University of Oulu,Oulu,Finland

3Department of Theoretical Chemistry,Materials Science Centre,Rijksuniversiteit Groningen,Groningen,The Netherlands

ABSTRACT Molecular dynamics simulation techniques together with time-dependent density functional theory calculations have been used to investigate the effect of photon absorption by a 4-hydroxy-cinnamic acid chromophore on the struc-tural properties of the photoactive yellow protein (PYP)from Ectothiorodospira halophila.The calcu-lations suggest that the protein not only modifies the absorption spectrum of the chromophore but also regulates the subsequent isomerization of the chromophore by stabilizing the isomerization tran-sition state.Although signaling from PYP is thought to involve partial unfolding of the protein,the me-chanical effects accompanying isomerization do not appear to directly destabilize the protein.Proteins 2002;48:202–211.©2002Wiley-Liss,Inc.

Key words:photoactive yellow protein;signal trans-duction;photon absorption;photo-

isomerization;molecular dynamics;

time-dependent DFT

INTRODUCTION Ectothiorodospira halophila,a small salt-tolerant bacte-rium,avoids excessive exposure to harmful ultraviolet radiation by detecting blue light and moving in a direction opposite to the gradient.1The cytoplasm of E.halophila contains a small water-soluble protein,named photoactive yellow protein(PYP),that has an absorption maximum that closely matches the action spectrum for the negative phototaxis.Therefore,PYP has been proposed to be the primary photoreceptor for this biologic process.PYP also serves as a structural prototype for the widely distributed Per–Arnt–Sim(PAS)class of signal transduction pro-teins.2

PYP contains a4-hydroxy-cinnamic acid(or p-coumaric acid)chromophore linked covalently to the␥-sulfur of Cys69via a thioester bond(Fig.1).3The chromophore is completely buried within a hydrophobic pocket with no exposure to solvent.The apolar environment lowers the p K

a

of the chromophore’s phenolic group to such an extent that this group is deprotonated at pHϭ7.0.The resulting net negative charge on the phenolic oxygen atom is stabi-lized by a hydrogen bonding network formed between this oxygen atom and the hydroxyl groups of three nearby amino acid side-chains[Tyr42,Glu46,and Thr50;Fig.

1.(b)].4The negative charge is further stabilized by charge delocalization over the chromophore via the conjugated p

z orbitals.Due to its interactions with nearby amino acid residues,the bound chromophore absorbs photons of blue light(␭

maxϭ446nm),giving the protein its characteristic yellow color.

Upon absorbing a photon,PYP enters a reversible photocycle,depicted in Fig.2.At room temperature,four distinct intermediate states can be identified along the cycle.The state described in the previous paragraph is the resting or ground state in the cycle,denoted by pG.In this state,the protein can be activated by absorbing a photon. The absorption of a photon triggers a trans-to-cis isomeriza-tion of the double bond in the chromophore[bond b,Fig. 1(a)]within a few nanoseconds.5,6Infrared spectroscopy has shown that during the isomerization process the hydrogen-bonding network,which stabilized the deproto-nated chromophore in the ground state,7remains intact. After photoisomerization,the protein is in the red-shifted state,denoted by pR.This name reflects the observed redshift in the absorption frequencies with respect to the ground state.

Over a period of microseconds,the protein then partially unfolds,leading to the blue-shifted state,in which the unfolding is at a maximum.8,9The blue-shifted state, denoted by pB,is believed to be the activated or signaling state.During the unfolding process,the phenolate group of the chromophore becomes protonated.This leads to a higher electronic stability of the chromophore and hence to a blueshift in the absorption frequencies.The precise order of the events that lead to this intermediate state is not known.

After the blue-shifted state,the protein completes the final stage of the cycle.The protein slowly refolds,the chromophore reisomerizes and loses its proton,and the system is restored to its equilibrium state,the ground

*Correspondence to:Alan E.Mark,Department of Biophysical Chemistry,Groningen Biomolecular Sciences and Biotechnology Insti-tute,Rijks universiteit Groningen,Nijenborgh4,9747AG Groningen, The Netherlands.E-mail:a.e.mark@chem.rug.nl

Received2August2001;Accepted15February2002 PROTEINS:Structure,Function,and Genetics48:202–211(2002)

©2002WILEY-LISS,INC.