Photochemistry and Photobiology, 1999, 70(6): 949-956 zyxwvu Studies on Pyrylretinal Analogues of Bacteriorhodopsin Joydip Das', Rosalie K. Crouch*', Rajnl Govindjee2,Sergei Balashov2 and Thomas Ebrey2 'Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA and *Department of Physiology and Biophysics, University of Illinois, Urbana, zyxwv IL, USA Received 3 June 1999; accepted 13 September 1999 ABSTRACT zyxwvutsr The retinal analogues 3-methyl-5-(l-pyryl)-2E,4E-penta- dienal ( 1 ) and 3,7-dimethyl-9-( 1 -pyryl)-2E,4E,6E,8E-non- atetraenal (2), which contain the tetra aromatic pyryl system, have been synthesized and characterized in order to examine the effect of the extended ring system on the binding capabilities and the function of bacteriorhodop- sin (bR). The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also ex- amined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retin- als formed pigments with all three bacterioopsins, and these pigments were found to have absorption maxima in the range 498-516 nm. All the analogue pigments showed activity as proton pumps. The pigment formed from wild-type apoprotein bR with 1 (with the shortened polyene side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton up- take. Extending the polyene side chain to the length iden- tical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and 0 intermediates at 435 nm and 650 nm, respectively. This pigment shows both fast and slow proton release at pH 7, suggesting that the pK, of the proton release group (in the M-state) is higher in this pigment compared to native bR. Hydrogen azide ions were found to accelerate the rise and decay of the 0 intermediate at neutral pH in pyryl 2 pigment. The pigments formed between 2 and E194Q and E204Q showed proton-pumping behavior similar to pigments formed with the native retinal, suggesting that the size of the chromophore ring does not alter the protein confor- mation at these sites. INTRODUCTION Bacteriorhodopsin (bR)t is the transmembrane photorecep- tor protein in the purple membrane of Halobacterium zyxwvut sali- narium and functions as a light-driven proton pump (1). The *To whom correspondence should be addressed at: College of Grad- uate Studies, Medical University of South Carolina, 96 Jonathon Lucas Street, Suite 601, Charleston, SC 29425, USA. Fax: zyxwvuts 843- 792-6590; e-mail: crouchrk@musc.edu ?Abbreviations: bR, bacteriorhodopsin; DIBAL, diisobutylalumi- num hydride; DQ, double quantum filtered; OS, opsin shift; PSB, protonated Schiff base; SB, Schiff base; THF. tetrahydrofuran. zyxwvut $5.00+0.00 zyxwvutsrq 0 1999 American Society for Photobiology 003 1-8655/99 chromophore of this pigment is retinal (6), which is attached to the e-amino group of lys-216 of the apoprotein zy via a pro- tonated Schiff base linkage (2). Upon absorption of light, the protein undergoes a photocycle having several intermediates with distinct spectral characteristicsand translocates a proton from the cytoplasmic side to the extracellular side of the membrane (2,3). In recent years much effort has been di- rected toward understanding the unique spectral and photo- chemical properties of bR. In this context a large number of synthetic chromophore analogues have been incorporated into the binding site of bR and the results were analyzed in terms of chromophore-protein interactions (43). These stud- ies have provided significant information on the steric and electronic properties of the chromophore binding site. The trimethyl cyclohexenyl ring of the native retinal (6) has been replaced by the phenyl (6-8), naphthyl (9-12) and phenan- thryl (13) rings (Fig. l), and it was concluded that the ring binding site is unrestrictive, at least to these systems. We have extended these studies to the pyryl retinal derivatives containing the tetra aromatic ring system. More recently, research on the development of bR variants has also gained momentum due to the possibility of produc- ing proteins of enhanced performance for applications in molecular electronics (14,15). Bacteriorhodopsin analogues have been developed that contain chromophores that both alter the proteins' absorption characteristics while maintain- ing the photochemical activity and slowing the decay of the M intermediate (16-18). New derivatives of bR have been generated both by replacing the chromophore with retinal derivatives and by the substitution of amino acid residues that are crucial for the functional properties of bR. For ex- ample, D96N reconstituted with 4-keto retinal has been stud- ied in gelatin films and has been found to be advantageous as compared to the 4-keto wild-type (WT) bR (19). In a similar study, 14-F retinal derivatives of D96A, E204Q, R82Q and D85N were generated and the proton transfer mechanism examined (20). The limitations of the ring binding site of the chromo- phore in bR are still poorly understood. We therefore have synthesized the pyryl derivative in order to test the effect of straining the ring binding site by the addition of another aromatic ring. We report here the preparation and character- ization of bR derivatives consisting of the chromophores 3- methyl-5-( l-pyryl)-2E,4E-pentadienal (1) and 3,7-dimethyl- 9-( l-pyryl)-2E,4E,6E,8E-nonatetraenal (2) and the WT, E204Q and E194Q apomembranes. 949