1. Cryostat ~1.4K Excitation Intensity ~50 W cm -2 Max. Scan speed: 0.3 nm s -1 (~50cm -1 s -1 ) Acquisition time 10 ms per data point Result: nominal resolution ~0.5cm -1, spectral bandwidth of the laser 1 cm -1. Alpha-polypeptides found in the low-light LH2 Rps. palustris (nLC-MS): Mg B850 acetyl-carbonyl group B850 “  e ” 1 M---AILVHFAVLSNTTWFSKYWNG----KAAAIR- 53 “  d ” 1 M---VFLVHFAVLTHTTWVAKFMNG----KAAAIES 54 Low-light LH2 complexes from Rps. palustris 2.1.6. have multiple types of αβ-apoproteins in individual rings: A single-molecule study Tatas H.P. Brotosudarmo 1 (t.brotosudarmo.1@research.gla.ac.uk), Alastair T. Gardiner 1, Vladimíra Moulisová 1, Ralf Kunz 2, Jürgen Köhler 2 * and Richard J. Cogdell 1 * 1 Department of Biochemistry & Molecular Biology, Faculty of Biomedical & Life Sciences, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK 2 Experimental Physics IV and Bayreuth Institute for Macromolecular Research, Universität Bayreuth, Universitätstrasse 30, D-95440 Bayreuth, Germany *To whom correspondence should be addressed. Abstract – Rps. palustris strain 2.1.6 belongs to the group of purple bacteria, which have the ability to produce LH2 antenna complexes that have unusual absorption spectra, when the bacteria are grown at low light intensity. This ability is often related to the presence of the multiple antenna apoproteins. This raises the interesting question as to whether it is possible to have LH2 rings in which there are multiple types of αβ-apoproteins present in the same ring. This possibility has been tested using single-molecule spectroscopy. Here we report, for the first time, the existence of individual low-light LH2 complexes that display mixed spectroscopic properties that indicate the presence of B800, “B820” and “B850”-like BChls. The arrangement of the BChla molecules in the mixed B820/850 ring can be modeled by 9 αβ BChls dimers, where 6 α-bound BChl a molecules have B820-like site energies and the remaining BChl a molecules have B850-like site energies. Furthermore, the experimental data can only be satisfactorily modeled when these 6 α-bound B820-like BChl a molecules are symmetrically distributed on the 3-fold axes in the ring. It is also clear from the measured single-molecule spectra that the main type of energetic disorder in the mixed B820/850 ring is diagonal. Light-harvesting 2 (LH2) Rps. palustris Figure 1. 77K Absorption spectra (A) and circular dichroism spectra (B) of various LH2 mixtures from Rps. palustris grown at different light intensity. No high resolution X-ray structure yet Our recent 5.0 Å electron density map for lowlight LH2 suggested: 9-mers of αβ-polypeptide with 2 rings of BChl a molecules (weakly coupled B800 & tightly coupled B850) 77K CD suggested “820 species” in the tightly coupled ring. Mixture of α-polypeptide compositions in the LH2 antenna Conclusions 1.The existence of individual low-light LH2 complexes that display B800, “B820 “and “B850 “-like BChls 2.In a single-molecule BChls in the tightly-coupled ring have B850-like and B820-like site-energies 3.The arrangement of the BChls molecules in the mixed B820/850 ring can be modelled: 9 alpha-beta BChls dimers, where 6 alpha-bound BChls have B820-like site energy and the remaining BChl molecules have B850-like site-energy. 4.These 6 alpha-bound BChl molecules are distributed symmetrically about the 3-fold axes in the ring. 5.The main disorder is diagonal” Acknowledgement – This work was supported by BBSRC, UK and DFG, Germany. THPB and VM acknowledges the fellowship from the European Commission through the Human Potential Program (Marie-Curie RTN BIMORE, Grant No. MRTN-CT-2006-035859). We acknowledge Aaron Collin for the absorption and CD spectra at 77 K (Figure 1). We also thank to Paul Boehm for the help in single-molecule experiment. REFERENCES: Cogdell, R.J., Gall, A., and Köhler, J. (2006) The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecule to in vivo membranes. Q. Rev. Biophys., 39, 227-324 Evans, M.B., Hawthornthwaite, A.M., and Cogdell, R.J. (1990) Isolation and Characterisation of the different B800-850 Light- Harvesting Complexes from Low- and High-Light Grown Cell of Rhodopseudomonas palustris, strain 2.1.6, Biochem. Biophys. Acta. 1016, 71-76 Gall, A. and Robert, B. (1999) Characterization of the Different Peripheral Light-Harvesting Complexes from High- and Low- Light Grown Cells from Rhodopseudomonas palustris, Biochemistry 38, 5185-5190 Tadros, M.H. and Waterkamp, K. (1989) Multiple Copies of the Coding Regions for the Light-Harvesting B800-850 α- and β- Polypeptides are present in the Rhodopseudomonas palustris Genome, The EMBO Journal 8(5), 1303-1308 Hofmann, C., Aartsma, T.J. and Köhler, J. (2004) Energetic disorder and the B850-exciton states of individual light-harvesting 2 complexes from Rhodopseudomonas acidophila, Chem. Phys. Lett. 395, 373-378 Experimental setup Observed LT SM-spectra Results 9-mer and 8-mer models comparedExtended simulationsBest model Simulations Figure 2. Low-temperature (1.4 K) fluorescence-excitation spectra of LH2 complexes from LL Rps. palustris 2.1.6. The excitation intensity was 50 W cm-2. Figure 3A. Stack of 410 individual spectra recorded consecutively. Between two successive spectra the polarization of the incident radiation has been rotated by 6.4°. B Top: Fluorescence intensity of the four bands B1 - B4 marked by the arrows in the lower part as a function of the polarization of the incident radiation (dots) together with cos 2 -type functions (colored) fitted to the data. Bottom: Two fluorescence-excitation spectra from the stack that correspond to mutually orthogonal polarization of the excitation light. Figure 4. Information gathered from the SMS: 1. Distributions of the energetic separations  E observed between A) B1 and B2, B) B1 and B3, and C) B1 and B4. 2. Distributions of the mutual angles  between the transition-dipole moments that are associated with D) B1 and B2, E) B1 and B3, and F) B1 and B4. Figure 5. Examples for simulated absorption spectra for an individual LH2 complex (solid line). The spectra correspond to a single realisation of the disorder. The calculated energies of the exciton states have been dressed by a Lorentzian with a width of 4.3 cm-1 for the k= 0 state and a width of 127 cm-1 for all other exciton states (colored lines). The simulations A-C (left) are based on a nonamaric structure, the simulations D-F (right) are based on an octameric structure. The simulations vary with respect to the type of diagonal disorder: A, D: random diagonal disorder taken from a Gaussian distribution of width 160 cm-1 (FWHM). B, E: random diagonal disorder like for A, D and additionally correlated diagonal disorder. C, F: like B, E and additionally using B820 and B850 like randomly distributed site energies across the ring assembly Figure 6. Comparison of the energetic separations of the bands B1-B4 with the separations of the respective exciton states (left) and the mutual orientation of the associated transition-dipole moments (right) as predicted from Monte Carlo simulations for 2000 realisations of the disorder (squares) for (A) the model shown in Fig. 5E, (B) the model shown in Fig. 5F and (C) the model shown in Fig. 5C. Figure 7. Extended simulations based on the model shown in Fig. 5C, i.e. a nonameric structure with random- and correlated diagonal disorder and multipolypeptide composition. (A) A B820-like site- energy of E0 (  B820) = 12400 cm-1 was assigned to all  -bound-Bchl a molecules and a B850-like site-energy of E0 (  B850) = 12060 cm-1 was assigned to all  -bound-Bchl a molecules. (B) Refinement of the extended model by assigning the B820-like site energies to fewer  -bound-Bchl molecules. The lines in the top part of the figure indicate the B820 like (red line) and B850 like (black line) site energies. Figure 8. Top: Model structure that features 6  - bound 820-like Bchl a molecules (red lines) distributed in C3 symmetry around the ring. Bottom: Comparison of the experimental data with the results from Monte Carlo simulations (2000 realisations) for the nonameric structure as shown in the top part taking random- and correlated diagonal disorder into account (black squares). Previous studies on low-light LH2 Rps. palustris Scheuring et al. (2006) J Mol Biol. 385: 83 Hartigan et al. (2002) Biophys J. 82:963 de Ruijter et al. (2004) Biophys J. 87: 3413