Rhodopsin in nanodiscs has native membrane-like photointermediates

Hisao Tsukamoto, Istvan Szundi, James W. Lewis, David Farrens, David S. Kliger

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5086-5091
Number of pages6
JournalBiochemistry
Volume50
Issue number22
DOIs
StatePublished - Jun 7 2011

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Rhodopsin
Membranes
Detergents
Photoexcitation
Light scattering
Suspensions
Chemical activation
Light
Kinetics
Temperature
Time delay
Membrane Proteins
Optical properties
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tsukamoto, H., Szundi, I., Lewis, J. W., Farrens, D., & Kliger, D. S. (2011). Rhodopsin in nanodiscs has native membrane-like photointermediates. Biochemistry, 50(22), 5086-5091. https://doi.org/10.1021/bi200391a

Rhodopsin in nanodiscs has native membrane-like photointermediates. / Tsukamoto, Hisao; Szundi, Istvan; Lewis, James W.; Farrens, David; Kliger, David S.

In: Biochemistry, Vol. 50, No. 22, 07.06.2011, p. 5086-5091.

Research output: Contribution to journalArticle

Tsukamoto, H, Szundi, I, Lewis, JW, Farrens, D & Kliger, DS 2011, 'Rhodopsin in nanodiscs has native membrane-like photointermediates', Biochemistry, vol. 50, no. 22, pp. 5086-5091. https://doi.org/10.1021/bi200391a
Tsukamoto, Hisao ; Szundi, Istvan ; Lewis, James W. ; Farrens, David ; Kliger, David S. / Rhodopsin in nanodiscs has native membrane-like photointermediates. In: Biochemistry. 2011 ; Vol. 50, No. 22. pp. 5086-5091.
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