Lumi I → Lumi II: The Last Detergent Independent Process in Rhodopsin Photoexcitation

Epps, Jacqueline; Lewis, James W.; Szundi, István; Kliger, David S.

doi:10.1111/j.1751-1097.2006.tb09796.x

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…Time-resolved absorbance changes from 300 to 700 nm were measured at 20 °C using an apparatus described previously (11). Instead of the dye laser excitation used there, samples were excited directly with the third harmonic of the Nd:YAG laser.…”

Section: Methodsmentioning

confidence: 99%

Schiff Base Protonation Changes in Siberian Hamster Ultraviolet Cone Pigment Photointermediates

et al. 2012

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Molecular structure and function studies of vertebrate ultraviolet (UV) cone visual pigments are needed to understand the molecular evolution of these photoreceptors, which uniquely contain unprotonated Schiff base linkages between the 11-cis retinal chromophore and the opsin proteins. In this study, the Siberian hamster ultraviolet cone pigment (SHUV) was expressed and purified in an n-dodecyl-β-D-maltoside suspension for optical characterization. Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were made on the purified pigment at time delays from 30 ns to 4.64 seconds after photoexcitation using 7 ns pulses of 355 nm light. The resulting data were fit globally to a sum of exponential functions after noise reduction using singular value decomposition. Four exponentials best fit the data with lifetimes of 1.4 µs, 210 µs, 47 ms and 1 s. The first photointermediate species characterized here is an equilibrated mixture similar to the one formed after rhodopsin's Batho intermediate decays into equilibrium with its successor, BSI. The extremely large red shift of the SHUV Batho component relative to the pigment suggests that SHUV Batho has a protonated Schiff base and that the SHUV cone pigment itself has an unprotonated Schiff base. In contrast to SHUV Batho, the portion of the equilibrated mixture's spectrum corresponding to SHUV BSI is well fit by a model spectrum with an unprotonated Schiff base. The spectra of the next two photointermediate species revealed that they both have unprotonated Schiff bases and suggest they are analogous to rhodopsin's Lumi I and Lumi II species. After decay of SHUV Lumi II, correspondence with rhodopsin photointermediates breaks down and the next photointermediate, presumably including the G protein-activating species, is a mixture of protonated and unprotonated Schiff base photointermediate species.

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Section: Methodsmentioning

confidence: 99%

Schiff Base Protonation Changes in Siberian Hamster Ultraviolet Cone Pigment Photointermediates

et al. 2012

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“…Rhodopsin was prepared and solubilized using methods described previously (17). For measurements at pH 6.5, the rhodopsin pellet obtained after hypotonic washing was resuspended in 5% lauryl maltoside (LM), 10 mM MES, 100 mM NaCl adjusted to pH 6.5, and for measurements at pH 8.7, the hypotonically washed rhodopsin pellet was resuspended in 5% LM, 10 mM Tris, 100 mM NaCl adjusted to pH 8.7.…”

Section: Methodsmentioning

confidence: 99%

“…Time-resolved absorbance changes from 300 to 700 nm (A photolyzed (λ) - A unphotolyzed (λ)) were measured at temperatures 15 and 30 °C using an apparatus described previously (17). Absorbance difference spectra were collected at a series of delay times after the ~7 ns excitation pulse of 477 nm light (80 μJ/mm 2 ), beginning at 10 μs and ending at either 240 ms (15 °C) or 20.48 ms (30 °C).…”

Section: Methodsmentioning

confidence: 99%

Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates

et al. 2011

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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, 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 I480 ⇄ 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.

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“…A transient blue-shifted intermediate (BSI) cannot be trapped at low temperatures. Time-resolved UV–vis measurements revealed the existence of additional transient forms of Lumi (Lumi II), 570,571 and Meta I (Meta I 380 ; 572,573 Meta Ib). 574 The RSBH + remains protonated up through Meta I, probably due to the low p K a of the stabilizing counterion Glu113.…”

Section: Animal Rhodopsinsmentioning

confidence: 99%

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms

et al. 2013

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Lumi I → Lumi II: The Last Detergent Independent Process in Rhodopsin Photoexcitation

Cited by 5 publications

References 28 publications

Schiff Base Protonation Changes in Siberian Hamster Ultraviolet Cone Pigment Photointermediates

Schiff Base Protonation Changes in Siberian Hamster Ultraviolet Cone Pigment Photointermediates

Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms

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