Does the Reconstitution of RC-LH1 Complexes fromRhodopseudomonas acidophilaStrain 10050 into a Phospholipid Bilayer Yield the Optimum Environment for Optical Spectroscopy?

Abstract

We have investigated reaction-center light-harvesting 1 (RC-LH1) complexes from Rhodopseudomonas (Rps.) acidophila in detergent buffer solution and reconstituted into a phospholipid bilayer and compared the results with the outcome of an earlier study conducted on RC-LH1 immobilized in polyvinyl alcohol (PVA). The aim of this study was to test whether the immobilization of the complexes in a PVA matrix might lead to a deterioration of the proteins and thereby limit the accessible information that can be obtained from optical spectroscopy. It has been found that the complexes dissolved in a detergent buffer solution are subject to fast spectral dynamics preventing any meaningful application of single-molecule spectroscopy. In contrast, for the bilayer samples it is revealed that the reconstitution process results in a significantly larger fraction of broken complexes with respect to the preparation of the complexes in a PVA film. Moreover, we find that for the intact complexes the statistics of the key spectral features, such as the spectral separations of the bands and the mutual orientation of their transition-dipole moments, show no variation dependent on using either a bilayer or PVA as a matrix. Given the additional effort involved in the reconstitution process, the lower amount of intact RC-LH1 complexes and, concerning the decisive spectral details, the identical results with respect to embedding the complexes in a PVA matrix, we come to the conclusion that the immobilization of these proteins in a PVA matrix is a good choice for conducting low-temperature experiments on individual light-harvesting complexes.

Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

“…Recently, however, the possibility of a static deformation of LH2 complexes due to the PVA matrix has been discussed in (36). To verify whether embedding the LH2 complexes in a PVA matrix deforms the structure of these antenna complexes and, therefore, introduces, per se, significant changes in their spectroscopic behavior, we reconstituted LH2 and RC-LH1 into a lipid bilayer and compared the spectra with those from complexes that were embedded in PVA (30,67). These studies revealed that at least for the LH complexes from purple bacteria, PVA is a good choice as a matrix that does not influence the conclusions drawn about the excitonic states in those pigment-protein complexes.…”

Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy

“…Since in the ABEL trap the feedback signal that compensates for the diffusion of the protein is generated from the emission of the sample, the observation times are typically limited to some ten seconds before the particle crosses over to the off state and gets lost from the trap. It is, however, possible to extend the observation time by immobilizing LH2 in a polymer film under conditions where this does not significantly perturb the structure of the protein 31,32 . This approach has enabled us to follow the dynamics of the LH2 fluorescence intermittency over an unprecedented nine orders of magnitude in time.…”

Contribution of low-temperature single-molecule techniques to structural issues of pigment–protein complexes from photosynthetic purple bacteria

“…acidophila immobilized in DDM/PVA (grey) and reconstituted into a DOPC lipid bilayer (red). Data from [64].…”

“…acidophila into a phospholipid rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20170680 bilayer and compared the results with those obtained from LH complexes immobilized in a polymer [64,65]. Of course, singlemolecule spectroscopy cannot solve structures but it allows the distributions of spectral parameters to be obtained rather than their moments only, which is what one would obtain from (sub-) ensemble techniques.…”