Oriented Insertion of ESR-Containing Hybrid Proteins in Proteoliposomes

Petrovskaya, L.E.; Lukashev, E.P.; Mamedov, M.D.; Kryukova, E.A.; Balashov, S.P.; Dolgikh, D.A.; Rubin, A.B.; Kirpichnikov, M.P.; Siletsky, S.A.

Авторы: Petrovskaya Lada E., Lukashev Evgeniy P., Mamedov Mahir D., Kryukova Elena A., Balashov Sergei P., Dolgikh Dmitry A., Rubin Andrei B., Kirpichnikov Mikhail P., Siletsky Sergey A.
Журнал: International Journal of Molecular Sciences
Том: 24
Номер: 8
Год издания: 2023
Издательство: MDPI
Местоположение издательства: Basel, Switzerland
Первая страница: 7369
Последняя страница: 7387
DOI: 10.3390/ijms24087369
Аннотация: Microbial rhodopsins comprise a diverse family of retinal-containing membrane proteins that convert absorbed light energy to transmembrane ion transport or sensory signals. Incorporation of these proteins in proteoliposomes allows their properties to be studied in a native-like environment; however, unidirectional protein orientation in the artificial membranes is rarely observed. We aimed to obtain proteoliposomes with unidirectional orientation using a proton-pumping retinal protein from Exiguobacterium sibiricum, ESR, as a model. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized. The photocycle of the hybrid proteins incorporated in proteoliposomes demonstrated a higher pKa of the M state accumulation compared to that of the wild-type ESR. Large negative electrogenic phases and an increase in the relative amplitude of kinetic components in the microsecond time range in the kinetics of membrane potential generation of ESR-Cherry and ESR-Trx indicate a decrease in the efficiency of transmembrane proton transport. On the contrary, Caf-ESR demonstrates a native-like kinetics of membrane potential generation and the corresponding electrogenic stages. Our experiments show that the hybrid with Caf1M promotes the unidirectional orientation of ESR in proteoliposomes.
Добавил в систему: Силецкий Сергей Алексеевич

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Oriented Insertion of ESR-Containing Hybrid Proteins in Proteoliposomesстатья

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