Fluorescence Energy Donor Research Articles

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb 3+ chelated to dipicolinate and the acceptor was the 11- cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R 0 value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.

The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS § § Abbreviations used: PABS, p-phenylazobenzene-sulfonyl; DPBS, 4-(diethylamino)-phenylazobenzene-4-sulfonyl. gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes.