Article

Assembly of the major light-harvesting complex II in lipid nanodiscs.

Section of Biophysics, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
Biophysical Journal (Impact Factor: 3.83). 11/2011; 101(10):2507-15. DOI: 10.1016/j.bpj.2011.09.055
Source: PubMed

ABSTRACT Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q(y) region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.

0 Followers
 · 
151 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The photocycle and vibrational dynamics of bacteriorhodopsin in a lipid nanodisc microenvironment have been studied by steady-state and time-resolved spectroscopies. Linear absorption and circular dichroism indicate that the nanodiscs do not perturb the structure of the retinal binding pocket, while transient absorption and flash photolysis measurements show that the photocycle which underlies proton pumping is unchanged from that in the native purple membranes. Vibrational dynamics during the initial photointermediate formation are subsequently studied by ultrafast degenerate transient absorption spectroscopy, where the low scattering afforded by the lipid nanodisc microenvironment allows for unambiguous assignment of ground and excited state nuclear dynamics through Fourier filtering of frequency regions of interest and subsequent time domain analysis of the retrieved vibrational dynamics. Canonical ground state oscillations corresponding to high frequency ethylenic and C-C stretches, methyl rocks, and hydrogen out-of-plane wags are retrieved, while large amplitude, short-lived vibrations are recovered predominantly in the frequency region associated with out-of-plane dynamics and low frequency torsional modes implicated in isomerization.
    Physical Chemistry Chemical Physics 08/2014; 16(39). DOI:10.1039/C4CP01826E · 4.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, since detergents commonly affect their structure and/or activity. Here we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the thus purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the thus obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the "light-harvesting conformation", whereas the "quenched conformation" is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30 % shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.
    Journal of Membrane Biology 08/2014; 247(9-10). DOI:10.1007/s00232-014-9712-6 · 2.17 Impact Factor

Full-text (2 Sources)

Download
46 Downloads
Available from
May 21, 2014