Binding of, and Energy-Transfer Studies from Retinal to, Organic Cations in Regenerated Reduced Bacteriorhodopsin

Abstract

Regenerated R b R from deionized R b R (dIRbR) and fluorescent 3,6-diaminoacridine cation (DAA+) is studied as a function of the cation to dIRbR ratio. Using the fluorescence intensity of DAA+, a Scatchard plot was drawn from which two DAA+ are found to be strongly bound while more than three are found to be weakly bound. The retinal in reduced bacteriorhodopsin (RbR) is fluorescent. The fluorescence intensity and decay of the retinal are examined under conditions where there are two strongly bound DAA+ per b R molecule.Assuming that energy transfer between the excited state of the retinal and the DAA+ is responsible for the observed shortening of the retinal excited-state lifetime, the Forster model was used to estimate a donoracceptor distance of 514 A. A discussion of the possible location of the DAA+ with respect to the retinal is given. A comparison between the organic and metal cation binding is made, and a discussion of the possible location of the metal cations in b R is presented.

Location of a Cation-Binding Site in the Loop between Helices F and G of Bacteriorhodopsin as Studied by 13C NMR

“…In this connection, caution should be used in referring to some observed phenomena as cation binding, such as highaffinity and low-affinity bindings based on Ca 2ϩ -selective electrode studies (Zhang et al, 1992Yang and El-Sayed, 1995;Yoo et al, 1995), spectroscopic titration (Ariki and Lanyi, 1986), electron spin resonance (Dunach et al, 1987), radioactive 45 Ca 2ϩ (Dunach et al, 1988b), fluorescence intensity of Ru(bpy) 3 2ϩ (Wu and El-Sayed, 1994a), 3,6-diaminoacridine cation (DAA ϩ ) (Wu and El-Sayed, 1994b), and Eu 3ϩ (Sweetman and El-Sayed, 1991), because there is a possibility that some of these data may be related to cationinduced pH changes. This is because the cation-binding sites in the deionized blue form may be different from those in the purple form.…”

The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin

“…This together with the observation that the strong binding site for Eu3+ is found to have only one Eu3+ ion suggests that the strong binding site must be near retinal, and thus its fluorescence is quenched and not seen in the emission studies (Sweetman and El-Sayed, 1991). The energy transfer studies between the retinal and different probing molecules such as Ru(bpy)32+ (Wu and El-Sayed, 1994a) and DAA+ (3,6,diaminoacridine) (Wu and El-Sayed, 1994b) again support the finding that at least one or two metal ions are located inside the protein not far from the retinal pocket. An alternative way of looking at Ca2+ binding is that some of metal ions are on the surface and others are inside the protein.…”