Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Abstract

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.

Nanodiscs in Membrane Biochemistry and Biophysics

“…193 Incorporation was performed under conditions yielding monomeric bR in Nanodiscs, which was confirmed by CD spectroscopy. Kinetics that follow the recovery of the 560 nm absorbance band after excitation at 532 nm were significantly faster when the DOPG or DMPG lipid fraction was 50% or higher, indicating an important contribution of anionic lipids in the protonation of the Schiff base.…”

“…420 Thus, the presence of negatively charged lipids is essential for fully functional signaling of nonvisual GPCR as well as for rhodopsins. 193 …”

Highly Efficient Transfer of 7TM Membrane Protein from Native Membrane to Covalently Circularized Nanodisc

“…The steady-state absorption spectrum of PMND showed a characteristic band at 563 nm after light adaptation. When compared to the absorption spectrum of solubilized bR in Triton X-100 at 553 nm 41 and the monomeric bR in synthetic lipid nanodiscs at 560 ± 1 nm 8 , PMND displayed a much smaller spectral shift from the native trimeric bR in PM at 568 nm (Figs 3C and S6 ) 41 , 42 . This suggests that the tertiary structure and lipid environment of bR in PMND is even closer to bR in PM compared to bR in synthetic lipid nanodiscs.…”

“…However, the native membrane bilayer and its lipid composition have unique properties that have specific effects on the functions, stability and/or folding of membrane proteins 11 , 12 . Therefore, despite providing a near-native membrane bilayer, the lipids that make up the nanodisc can still influence the lipid-protein interaction and alter the behavior of the membrane protein 8 , 13 , 14 .…”

“…In particular, the transition to the M state is known to be related to the deprotonation of the Schiff base and the subsequently release of the proton to the extracellular side, while the O state indicates the relaxation of the retinal before the next photocycle begins 27 , 28 . bR has previously been reported to incorporate into lipid nanodiscs; however, the studies were done using synthetic lipids and including solubilizing bR with detergent before nanodisc assembly 5 , 8 , 9 , 29 , 30 . By observing these photocycle intermediates initiated by a pulsed laser, we showed that the function and activity of membrane protein embedded in the nanodisc, assembled directly from the native membrane, were able to be maintained.…”