Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

Improved Isolation Procedures for the Purple Membrane of Halobacterium Halobium

Techniques for purifying the purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromoprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.0.³ The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Physicochemical characterization of bovine retinal arrestin

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.     

Characterization of Langmuir-Blodgett films of rhodopsin: thermal stability studies.

Two-dimensional close packing of purified bovine rhodopsin, made by the Langmuir-Blodgett technique, was characterized by small angle x-ray scattering and nanogravimetric measurements. The area occupied by a molecule of rhodopsin in the film was approximately 1100 Angstrum2 and the periodicity of the layers resulted in 59 Angstrum. The circular dichroism measurements showed that bleached rhodopsin in Langmuir-Blodgett film had high thermal stability, in fact, reaching a temperature of 150 degrees C without a loss of the secondary structure. Moreover, when the film was made up in the dark, rhodopsin maintained its stability up to at least 200 degrees C and its characteristic absorbance peak at 500 nm up to about 90 degrees C.     

Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin

The role of negatively charged residues in tuning the absorbance spectrum of bovine rhodopsin has been tested by mutating each aspartate and glutamate to asparagine and glutamine, respectively. Previous work demonstrated that aspartate83, glutamate122, and glutamate134 can be replaced by neutral residues with little or no effect on the absorbance spectrum of the resulting pigment [Nathans, J. (1990) Biochemistry 29, 937-942]. With one exception, mutations at the remaining 19 aspartate and glutamate residues result in very nearly wild-type absorbance spectra. The exception is glutamate113: mutation to glutamine causes the pigment to absorb at 380 nm, reflecting deprotonation of the retinylidene Schiff's base. Upon addition of either chloride, bromide, or iodide, the absorbance rapidly shifts to 495, 498, or 504.5 nm, respectively, reflecting protonation of the Schiff's base. The progressive red shift observed upon addition of halides with larger atomic radii strongly suggests that halides are serving as the Schiff's base counterion. Halides have no effect on the absorbance spectrum of wild-type rhodopsin. I infer, therefore, that glutamate113 is the retinylidene Schiff's base counterion in wild-type rhodopsin. Sakmar et al. [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8309-8313] and Zhukovsky and Oprian [(1989) Science 246, 928-930] have arrived at the same conclusion based upon a related series of experiments. These data support a model in which spectral tuning in bovine rhodopsin results from interactions between the polyene chain of 11-cis-retinal and uncharged amino acids in the binding pocket.     

On the visual pigments of deep-sea fish

The retinal visual pigments of 52 species of deep-sea fish were measured by partial bleaching of detergent extracts. The retinae of 45 species contained only a single rhodopsin with maximum absorbance (λ_max) at a wavelength between 474 and 490 nm, matching both the region of highest intensity downwelling sunlight and the maximum emission of most deep-sea bioluminescence. Seven species were shown to have more than one visual pigment within their retinae and these had λ_max values that generally fell outside the usual range. One of these, Bonapartia pedaliota , was particularly interesting as, unlike most such multipigment species, it had one rhodopsin and one porphyropsin pigment, apparently based on different opsins. The relative proportions of the visual pigments in the seven multipigment species are presented.     

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees C. The crystalline environment favored the Meta I(480) photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 degrees C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I(480) decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII(a)) forms in 2D rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.     

pH dependence of photolysis intermediates in the photoactivation of rhodopsin mutant E113Q

Glutamic acid at position 113 in bovine rhodopsin ionizes to form the counterion to the protonated Schiff base (PSB), which links the 11-cis-retinylidene chromophore to opsin. Photoactivation of rhodopsin requires both Schiff base deprotonation and neutralization of Glu-113. To better understand the role of electrostatic interactions in receptor photoactivation, absorbance difference spectra were collected at time delays from 30 ns to 690 ms after photolysis of rhodopsin mutant E113Q solubilized in dodecyl maltoside at different pH values at 20 degrees C. The PSB form (pH 5. 5, lambda(max) = 496 nm) and the unprotonated Schiff base form (pH 8. 2, lambda(max) = 384 nm) of E113Q rhodopsin were excited using 477 nm or 355 nm light, respectively. Early photointermediates of both forms of E113Q were qualitatively similar to those of wild-type rhodopsin. In particular, early photoproducts with spectral shifts to longer wavelengths analogous to wild-type bathorhodopsin were seen. In the case of the basic form of E113Q, the absorption maximum of this intermediate was at 408 nm. These results suggest that steric interaction between the retinylidene chromophore and opsin, rather than charge separation, plays the dominant role in energy storage in bathorhodopsin. After lumirhodopsin, instead of deprotonating to form metarhodopsin I(380) on the submillisecond time scale as is the case for wild type, the acidic form of E113Q produced metarhodopsin I(480), which decayed very slowly (exponential lifetime = 12 ms). These results show that Glu-113 must be present for efficient deprotonation of the Schiff base and rapid visual transduction in vertebrate visual pigments.     

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.     

Purification of two forms of kanamycin acetyltransferase from Escherichia coli

Kanamycin acetyltransferase acylates aminoglycoside antibiotics using acetyl-CoA, and thereby conveys bacterial resistance to several clinically important antibiotics, notably amikacin. The enzyme was quantitatively and reproducibly released from Escherichia coli W677 harboring plasmid pMH67 by a modified osmotic shock procedure (bacterial cells are incubated overnight in sucrose and again without sucrose before onset of osmotic shock). The enzyme was purified by dye-ligand chromatography on Affi-Gel Blue in addition to antibiotic affinity chromatography on neomycin-Sepharose-4B. The activity did not increase with subsequent chromatography on ion-exchange, hydrophobic, or molecular-exclusion gels. However, both dye-ligand and molecular-exclusion chromatography, as well as disc-gel electrophoresis, separated the purified enzyme equally into two active protein fractions. Based on the more active of the two forms, the purification was 112-fold with a specific activity of 1.9 IU/mg. The less-active form has an unusual absorbance spectrum, with a maximum near 255 nm, which cannot be explained by the amino acid composition. Chromatography of this form alone regenerated both forms, suggesting that the enzyme is noncovalently conjugated to an uncharged chromophore, such as a lipid. The purified enzyme has a very sharp pH optimum at 5.5 with a plateau on the alkaline side, but is most stable between pH 8.5 and 9.5. Data from electrophoresis in the presence of sodium dodecyl sulfate and gel-filtration on Ultrogel AcA 44 are consistent with a tetrameric protein of 60-70,000 Da.     

Euphausiid visual pigments. The rhodopsins of Euphausia superba and Meganyctiphanes norvegica (Crustacea, Euphausiacea)

The rhabdoms of Euphausia superba contain one digitonin-extractable rhodopsin, lambda max 485 nm. The rhodopsin undergoes unusual pH- dependent spectral changes: above neutrality, the absorbance decreases progressively at 485 nm and rises near 370 nm. This change is reversible and appears to reflect an equilibrium between a protonated and an unprotonated form of the rhodopsin Schiff-base linkage. Near neutral pH and at 10 degrees C, the rhodopsin is partiaLly converted by 420-nm light to a stable 493-nm metarhodopsin. The metarhodopsin is partially photoconverted to rhodopsin by long-wavelength light in the absence of NH2OH; in the presence of NH2OH, it is slowly converted to retinal oxime and opsin. The rhodopsin of Meganyctiphanes norvegica measured in fresh rhabdoms by microspectrophotometry has properties very similar to those of the extracted rhodopsin of E. superba. Its lambda max is 488 nm and it is partially photoconverted by short wavelength irradiation to a stable photoconvertible metarhodopsin similar to that of E. superba. In the presence of light and NH2OH, the M. norvegica metarhodopsin is converted to retinal oxime and opsin. Our results indicate that previous determinations of euphausiid rhodopsin absorbance spectra were incorrect because of accessory pigment contamination.     

In vitro identification of rhodopsin in the green alga Chlamydomonas

The unicellular alga Chlamydomonas can detect both intensity and direction of the ambient light and adjust its swimming speed and direction accordingly. On the basis of physiological experiments, the functional photoreceptor for this visual process has recently shown to be a rhodopsin. We here report the in vitro identification of endogenous retinal and a rhodopsin in Chlamydomonas cell extracts and purified membrane preparations. The rhodopsin absorption spectrum has fine structure with the maximum at 495 nm and matches the action spectra for the behavioral light responses. The rhodopsin can be bleached and subsequently reconstituted with exogenous retinal. Labeling with [3H]retinal occurs in the final preparation only with a single protein with a molecular weight of 32,000. We conclude that this protein is the visual photoreceptor in Chlamydomonas.     

Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin

A water-soluble yellow protein, previously discovered in the purple photosynthetic bacterium Ectothiorhodospira halophila, contains a chromophore which has an absorbance maximum at 446 nm. The protein is now shown to be photoactive. A pulse of 445-nm laser light caused the 446-nm peak to be partially bleached and red-shifted in a time less than 1 microsecond. The intermediate thus formed was subsequently further bleached in the dark in a biphasic process occurring in approximately 20 ms. Finally, the absorbance of native protein was restored in a first-order process occurring over several seconds. These kinetic processes are remarkably similar to those of sensory rhodopsin from Halobacterium, and to a lesser extent bacteriorhodopsin and halorhodopsin; although these proteins are membrane-bound, they have absorbance maxima at about 570 nm, and they cycle more rapidly. In attempts to remove the chromophore for identification, it was found that a variety of methods of denaturation of the protein caused transient or permanent conversion to a form which has an absorbance maximum near 340 nm. Thus, by analogy to the rhodopsins, the absorption at 446 nm in the native protein appears to result from a 106-nm red shift of the chromophore induced by the protein. Acid denaturation followed by extraction with organic solvents established that the chromophore could be removed from the protein. It is not identical with all-trans-retinal and remains to be identified, although it could still be a related pigment. The E. halophila yellow protein has a circular dichroism spectrum which indicates little alpha-helical secondary structure (19%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.     

Reactivity of tryptophans in rhodopsin.

Oxidation with N-bromosuccinimide detects a total of about ten tryptophan residues in detergent-solubilized bovine rhodopsin. One of these tryptophans is more reactive in bleached than in unbleached rhodopsin, suggesting its involvement in the chromophore binding site. Oxidation of this residue is accompanied by loss of the 500nm. absorbance in unbleached rhodopsin. Similar experiments with bacteriorhodopsin are inconclusive.     

2'',3''-CYCLIC NADP AS A SUBSTRATE FOR 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE

Abstract– 2',3'-Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l-ethyl-(3-dimethylaminopropyl)-carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2',3'-cyclic nucleotides to 2'-phospho esters. The K _m for this substrate is the same as that for 2',3'-cyclic AMP (0.22 m m ) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme-substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31-fold purified enzyme) spectrophotometric coupled enzyme assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose-6-phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.     

Purification and Characterization of Banana Bunchy Top Virus

Abstract Banana bunchy top virus (BBTV) was successfully purified by a procedure which included pulverizing diseased tissues frozen in liquid nitrogen, clarification of the extract with chloroformbutanol, concentration of the virus by cycles of differential centrifugation, stirring and incubating at 4°C followed by a second cycle of differential centrifugation and sucrose density gradient centrifugation. The concentrations of BBTV obtained from leaf blades and midribs plus petioles were up to 0.34 and 0.56mg/kg tissue, respectively. The virus concentration was highest in diseased leaves collected from October to December, and lowest in those collected from June to September. BBTV is a luteovirus (particles 20– 22 nm in diameter), preparations of which have maximum absorbance at 257 nm, minimum absorbance at 240 nm and a A260/280 ratio of 1.46. The relative molecular mass (M_r) of BBTV coat protein subunit is 21,000, and that of its ssRNA is 2.0 × 10⁶.     

Phototaxis of Halobacterium salinarium requires a signalling complex of sensory rhodopsin I and its methyl-accepting transducer HtrI.

Sensory rhodopsin I (SRI) is a photoreceptor that mediates phototaxis in the archaeon Halobacterium salinarium. Receptor excitation is relayed to the motility system of the cell by the methyl-accepting transducer protein HtrI. In membranes prepared from cells that lack HtrI the absorbance difference maximum of SRI was shifted from 587 to 565 nm. The thermal decay of the metastable photocycle intermediate SRI373 was measured as time-dependent recovery of the absorbance at 590 nm. In the absence of HtrI the decay was slowed down by two orders of magnitude. When SRI was overproduced in cells that contained normal levels of HtrI, the decay of SRI373 was biexponential indicating two kinetically distinct species. Spectroscopic measurements on intact cells revealed the same effect of HtrI on SRI photocycling as found in isolated membranes. By transient exposure of membranes from wild-type cells to low ionic strength, the decay of SR373 was slowed to the same value found for untreated membranes in the absence of HtrI. In parallel, the absorbance difference maximum was shifted to 565 nm indicating that a physical interaction of HtrI and SRI had been irreversibly destroyed. Overproduction of SRI in the presence of wild-type amounts of HtrI did not increase the light sensitivity of the cells to orange light step down stimulation. It is concluded that SRI and HtrI form a stable complex in the cell membrane that signals to the flagellar motor and defines absorbance maximum, photocycling rate and photochemical efficiency of SRI.     

Rhodopsin in nanodiscs has native membrane-like photointermediates

Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I(480) ? Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.