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Abstract <p><b>Objective:</b> Bacteriorhodopsin (BR) from <i>Halobacterium salinarum</i> is a model membrane protein for which the details of structural transitions during its photocycle remain unclear. NMR spectroscopy offers unique insights into these dynamic processes but requires a suitable membrane-mimicking environment and efficient isotopic labeling strategies. This study aimed to develop optimized protocols for production of isotopically labeled BR, evaluate various membrane mimetics (micelles, bicelles, nanodiscs) for NMR studies, and stated bicelles as the most promising medium for high-resolution structural analysis. <b>Methods:</b> BR was expressed in <i>E. coli</i> BL21(DE3) as a Mistic fusion construct. Both uniformly (<sup>15</sup>N/<sup>13</sup>C/<sup>2</sup>H) and selectively (<sup>15</sup>N-Ala, <sup>15</sup>N-Val, <sup>15</sup>N-Leu) labeled proteins were produced using M9 minimal medium. Purification involved immobilized metal affinity and size-exclusion chromatography, followed by refolding into different membrane mimetics. Protein stability was assessed by UV–Vis spectroscopy (<i>A</i><sub>280</sub>/<i>A</i><sub>554</sub> ratio) and by color retention. NMR spectra (<sup>15</sup>N-TROSY) were recorded at 600–800 MHz, and the numbers of signals, line widths, and rotational correlation times were compared. H/D exchange strategy and selective labeling were used to separate the signals. <b>Results and Discussion:</b> Optimized expression yielded ~40–50 mg/L of functional, isotopically labeled BR. Among all tested media, bicelles based on either CHAPS or DH<sub>6</sub>PC provided the best combination of functional stability (maintained purple color for weeks) and spectral quality, outperforming DDM micelles and nanodiscs. Rotational correlation times for bicelles were ~30 ns, significantly smaller than those for nanodiscs (44 ns), explaining improved resolution. Negatively charged lipid (DMPG) further enhanced spectral dispersion. Selective <sup>15</sup>N-labeling of Ala, Val, and Leu, combined with H/D exchange strategy, enabled detection of over 300 spin systems. Thus, bicelles outperform both micelles (better stability) and nanodiscs (better spectral resolution). <b>Conclusions:</b> Bicelles, based on DH<sub>6</sub>PC and CHAPS, represent a highly promising membrane mimetic for NMR structural studies of BR. Combined with selective isotopic labeling and H/D exchange strategy, this approach enables high-quality spectra and long-term protein stability, paving the way for detailed structural and dynamic characterization of BR and related membrane proteins.</p>

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Bicelles as a Prospective Membrane Mimetic for NMR Structural Studies of Bacteriorhodopsin

  • 期刊论文

  • 发表日期：

    2026年5月12日

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  • M. V. Goncharuk,
  • M. A. Dubinnyi,
  • O. V. Nekrasova,
  • A. S. Anurieva,
  • P. K. Kuzmichev,
  • V. V. Chupin,
  • I. S. Okhrimenko,
  • V. I. Borshchevskiy,
  • E. V. Bocharov,
  • A. S. Arseniev,
  • S. A. Goncharuk

摘要

Abstract

Objective: Bacteriorhodopsin (BR) from Halobacterium salinarum is a model membrane protein for which the details of structural transitions during its photocycle remain unclear. NMR spectroscopy offers unique insights into these dynamic processes but requires a suitable membrane-mimicking environment and efficient isotopic labeling strategies. This study aimed to develop optimized protocols for production of isotopically labeled BR, evaluate various membrane mimetics (micelles, bicelles, nanodiscs) for NMR studies, and stated bicelles as the most promising medium for high-resolution structural analysis. Methods: BR was expressed in E. coli BL21(DE3) as a Mistic fusion construct. Both uniformly (15N/13C/2H) and selectively (15N-Ala, 15N-Val, 15N-Leu) labeled proteins were produced using M9 minimal medium. Purification involved immobilized metal affinity and size-exclusion chromatography, followed by refolding into different membrane mimetics. Protein stability was assessed by UV–Vis spectroscopy (A280/A554 ratio) and by color retention. NMR spectra (15N-TROSY) were recorded at 600–800 MHz, and the numbers of signals, line widths, and rotational correlation times were compared. H/D exchange strategy and selective labeling were used to separate the signals. Results and Discussion: Optimized expression yielded ~40–50 mg/L of functional, isotopically labeled BR. Among all tested media, bicelles based on either CHAPS or DH6PC provided the best combination of functional stability (maintained purple color for weeks) and spectral quality, outperforming DDM micelles and nanodiscs. Rotational correlation times for bicelles were ~30 ns, significantly smaller than those for nanodiscs (44 ns), explaining improved resolution. Negatively charged lipid (DMPG) further enhanced spectral dispersion. Selective 15N-labeling of Ala, Val, and Leu, combined with H/D exchange strategy, enabled detection of over 300 spin systems. Thus, bicelles outperform both micelles (better stability) and nanodiscs (better spectral resolution). Conclusions: Bicelles, based on DH6PC and CHAPS, represent a highly promising membrane mimetic for NMR structural studies of BR. Combined with selective isotopic labeling and H/D exchange strategy, this approach enables high-quality spectra and long-term protein stability, paving the way for detailed structural and dynamic characterization of BR and related membrane proteins.