Flexibility and Size Heterogeneity of the LH1 Light Harvesting Complex Revealed by Atomic Force Microscopy

Abstract

Previous electron microscopic studies of bacterial RCLH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the QB site of the RC to the quinone pool prior to reduction of the cytochrome bc1 complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (α1β1Bchl2) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process. Previous electron microscopic studies of bacterial RCLH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the QB site of the RC to the quinone pool prior to reduction of the cytochrome bc1 complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (α1β1Bchl2) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process. Photosynthetic organisms harvest light energy and convert it to a chemically useful form, using light harvesting (LH) 1The abbreviations used are: LH, light harvesting; RC, reaction center; Bchl, bacteriochlorophyll; AFM, atomic force microscope; EM, electron microscopy. and reaction center (RC) complexes. In the purple photosynthetic bacteria, the reaction center, which is the site of photochemistry, receives excitation energy from the light harvesting LH1 complex, which receives energy in turn from the LH2 complex (reviewed in Ref. 1Blankenship R.E. Molecular Mechanisms of Photosynthesis. Blackwell Science Ltd., Oxford2002: 61-82Crossref Google Scholar). The atomic structure of the Rhodopseudomonas acidophila LH2 complex (2McDermott G. Prince S.M. Freer A.A. Hawthornthwaite-Lawless A.M. Papiz M.Z. Cogdell R.J. Isaacs N.W. Nature. 1995; 374: 517-521Crossref Scopus (2509) Google Scholar) and the cryo-electron microscopy (EM) structure of the Rhodobacter sphaeroides complex (3Walz T. Jamieson S.J. Bowers C.M. Bullough P.A. Hunter C.N. J. Mol. Biol. 1998; 282: 833-845Crossref PubMed Scopus (249) Google Scholar) both revealed a circular arrangement of nine protomers, each consisting of an α and a β polypeptide. The LH2α polypeptides formed an inner ring, with the β ring outermost; in all, 27 bacteriochlorophyll (Bchl) molecules are bound to this structure (2McDermott G. Prince S.M. Freer A.A. Hawthornthwaite-Lawless A.M. Papiz M.Z. Cogdell R.J. Isaacs N.W. Nature. 1995; 374: 517-521Crossref Scopus (2509) Google Scholar). More recent work has established that LH1 surrounds the RC using an arrangement of 16 αβ protomers and 32 Bchls (4Jamieson S.J. Wang P. Qian P. Kirkland J.Y. Conroy M.J. Hunter C.N. Bullough P.A. EMBO J. 2002; 21: 3927-3935Crossref PubMed Scopus (131) Google Scholar) when there is no prulifloxacin PufX protein. In other bacteria, an LH1 ring of 15 αβ protomers, together with either PufX or a putative PufX homologue (W), form a continuous ring of protein around the RC (5Roszak A.W. Howard T.D. Southall J. Gardiner A.T. Law C.J. Isaacs N.W. Cogdell R.J. Science. 2003; 302: 1969-1972Crossref PubMed Scopus (600) Google Scholar, 6Siebert C.A. Qian P. Fotiadis D. Engel A. Hunter C.N. Bullough P.A. EMBO J. 2004; 23: 690-700Crossref PubMed Scopus (136) Google Scholar). The demonstration of both circular and elliptical forms of this LH1 complex provided evidence for its flexibility (4Jamieson S.J. Wang P. Qian P. Kirkland J.Y. Conroy M.J. Hunter C.N. Bullough P.A. EMBO J. 2002; 21: 3927-3935Crossref PubMed Scopus (131) Google Scholar). This property of the LH1 complex was suggested to be a significant factor in the export of quinol, the product of RC photochemistry, to the cytochrome bc1 complex (4Jamieson S.J. Wang P. Qian P. Kirkland J.Y. Conroy M.J. Hunter C.N. Bullough P.A. EMBO J. 2002; 21: 3927-3935Crossref PubMed Scopus (131) Google Scholar). For organisms such as Rhodospirillum rubrum, which assemble an (αβ)16 LH1 complex completely enclosing the RC, such flexibility would clearly be an essential feature of this LH complex and would imply a dynamic series of conformations in vivo. However, only the extremes of this dynamic population have been reported, and the flexibility hypothesis requires the imaging of several conformations at room temperature. For other photosynthetic bacteria such as R. sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas palustris other possibilities for quinol export became apparent when it was found that these bacteria assemble another polypeptide, PufX (W), into the LH1 complex. It was discovered that puf X- mutants were unable to photosynthesize (7Farchaus J.W. Oesterhelt D. EMBO J. 1989; 8: 47-54Crossref PubMed Google Scholar, 8Farchaus J.W. Barz W.P. Grunberg H. Oesterhelt D. EMBO J. 1992; 11: 2779-2788Crossref PubMed Scopus (72) Google Scholar); subsequently, such mutants were found to be impaired in their ability to shuttle quinones/quinols in and of the QB site of the RC (9Lilburn T.G. Beatty J.T. FEMS Microbiol. Lett. 1992; 100: 155-159Google Scholar, 10Barz W.P. Vermeglio A. Francia F. Venturoli G. Melandri B.A. Oesterhelt D. Biochemistry. 1995; 34: 15248-15258Crossref PubMed Scopus (101) Google Scholar). It was suggested that PufX forms part of the LH1 ring, providing a portal for quinol (11Cogdell R.J. Fyfe P.K. Barrett S.J. Prince S.M. Freer A.A. Isaacs N.W. McGlynn P. Hunter C.N. Photosynth. Res. 1996; 48: 55-63Crossref PubMed Scopus (87) Google Scholar). This concept would seem to be supported by work on mutants with an LH1 complex that is too small to completely surround the RC; these mutants can, therefore, allow free movement of quinones/quinols to the RC QB site, and so they are fully capable of photosynthetic growth, even in the absence of PufX (12McGlynn P. Hunter C.N. Jones M.R. FEBS Lett. 1994; 349: 349-353Crossref PubMed Scopus (66) Google Scholar). Studies on RC-LH1-PufX complexes in native membranes show that PufX causes a specific orientation of the RC, which is the likely cause of the long range organization of the core complexes (6Siebert C.A. Qian P. Fotiadis D. Engel A. Hunter C.N. Bullough P.A. EMBO J. 2004; 23: 690-700Crossref PubMed Scopus (136) Google Scholar, 13Frese R.N. Olsen J.D. Branvall R. Westerhuis W.H. Hunter C.N. van Grondelle R. Proc. Natl. Acad. Sci. U. A. PubMed Scopus (87) Google Scholar) and a in the core complex into in F. Wang J. Venturoli G. Melandri B.A. Barz W.P. Oesterhelt D. Biochemistry. PubMed Scopus Google Scholar, Francia F. J. Melandri B.A. D. J. Biol. 2004; PubMed Scopus Google Scholar). It is the LH1 complex of R. sphaeroides is and to there is no PufX a quinol In terms of LH1 we are at to the cryo-electron microscopy studies of that the R. LH1 complex lacking PufX can both circular and elliptical This is to the that the are in at and that LH1 molecules in be The of the atomic force microscope to the of with the to the for the and the to Previous studies have the of for imaging of the LH2 complexes of F. Engel A. EMBO J. PubMed Scopus Google R. sphaeroides J. D. J. Mol. Biol. 2003; PubMed Scopus Google and R. acidophila A. Cogdell R.J. J. 2003; PubMed Scopus Google Scholar). J. D. Proc. Natl. Acad. Sci. U. A. 2003; 100: PubMed Scopus Google Scholar) the imaging of native membranes of complexes by and were to show that LH1 formed an the RC, that it became circular of the In of the of in of the LH1 ring, it is to of the shapes and aggregation of which LH1 is This be with the LH2 complex, using the same J. D. J. Mol. Biol. 2003; PubMed Scopus Google Scholar) have large of the LH2 complex from R. sphaeroides by In we have forms of the LH2 complex, to in of the LH2 complex. In this it is that in can complexes into or on the form is or (4Jamieson S.J. Wang P. Qian P. Kirkland J.Y. Conroy M.J. Hunter C.N. Bullough P.A. EMBO J. 2002; 21: 3927-3935Crossref PubMed Scopus (131) Google Scholar). was used to of the RCLH1 complex of R. D. Qian P. A. Bullough P.A. Engel A. Hunter C.N. J. Biol. 2004; PubMed Scopus Google Scholar). It was that the complex may an shape in of in the a likely flexibility when in the This study a formed as a of the RC with the which of the possibilities for this complex. this in it is to of many LH1 complexes free of the RC; it is only that the inherent flexibility and even of LH1 be the RC, which sits within the LH1 ring, normally provides a barrier to substantial We have used to of LH1 and of LH2 of R. We that the LH2 have forms and the the complexes are In contrast, LH1 molecules a range of both ring and that elliptical, and even polygonal ring shapes as well as arcs and open these we that the LH1 ring is and we this property to the in which it is and to its within the photosynthetic were from the which was from and the lipid which was from were of or and R. sphaeroides used have been M.R. Olsen J.D. Hunter C.N. Mol. Microbiol. 1992; PubMed Scopus Google Scholar); M.R. Olsen J.D. Hunter C.N. Mol. Microbiol. 1992; PubMed Scopus Google Scholar); R. U. A. Scopus Google Scholar); M.R. Olsen J.D. Hunter C.N. Mol. Microbiol. 1992; PubMed Scopus Google Scholar). The was and the membranes were to the in Olsen J.D. Hunter C.N. Proc. Natl. Acad. Sci. U. A. 1994; PubMed Scopus Google Scholar). The was into the by were for the of the LH1 complex using a and a were in and membranes were as J.D. Hunter C.N. Proc. Natl. Acad. Sci. U. A. 1994; PubMed Scopus Google that in this work we used a temperature of and the membranes by at for the to prior to LH was and as in (3Walz T. Jamieson S.J. Bowers C.M. Bullough P.A. Hunter C.N. J. Mol. Biol. 1998; 282: 833-845Crossref PubMed Scopus (249) Google Scholar). For units of membrane were with of with at and a The was for an at a of with with a at The were by the of the at and these were used for using the lipid and from was as a for the For the of LH2 was by of the of for the was into the and for to bound The was the and of was to the For of LH1 the was and the was to The imaging used was the same as for the LH2 For the a was van J. Sci. Scopus Google Scholar). with a of force of and of from were were using in with a free of the was to of the free was and were at a of The of the was with from were by of the were by a and are in of from LH1 with LH2 LH1 complex no RC formed a population of between and in of a in is in The of the the was of the LH1 of LH1 of the of lipid bilayer with an the of In contrast, were found for the LH2 complex, of which to be from the by their the The of the two in the and 16 to and lipid were observed for LH2 with an of which well with the from the of LH1 LH2 were observed and of small The of the was in in of complexes are in and long range was observed for the for LH2 T. Jamieson S.J. Bowers C.M. Bullough P.A. Hunter C.N. J. Mol. Biol. 1998; 282: 833-845Crossref PubMed Scopus (249) Google and J. D. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). In LH1 rings formed small of well with a of This only be observed in of This was by a for a as by the of the of the complex The of LH1 rings were in the lipid membrane in the by a from the lipid to the of LH1 of In the this is It be that LH1 which to be circular in the well of in size. For the and in ring of and The of of LH1 by was by of the LH1 complexes in terms of ring with each at two rings and arcs were of an LH2 the of of of LH2 complexes was clearly and that this into a a and are in in it can be that of be found within of of the of a The LH2 two and two which are clearly of the complex the the from the lipid to the of LH2 was and in the orientation this was This has been from (3Walz T. Jamieson S.J. Bowers C.M. Bullough P.A. Hunter C.N. J. Mol. Biol. 1998; 282: 833-845Crossref PubMed Scopus (249) Google Scholar) and from studies on LH2 F. Engel A. EMBO J. PubMed Scopus Google Scholar, J. D. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). The between and is that in the there is no between adjacent rings in the in they are together show as the LH2 complexes are into the lipid bilayer in a We in this the between the LH2 molecules provides for of LH1 and LH2 a of LH1 complexes in a LH1 complexes a of in and In contrast, LH2 complexes no such and show of LH2 The was that under no even for of the LH2 complex the in and shape we observed for of the of or LH2 rings to be circular and of within The of and LH2 complexes the of the lipid bilayer was and This is in with the of J. D. J. Mol. Biol. 2003; PubMed Scopus Google Scholar) on the same complex. this we that rings to the of the LH2 complex. the of we found that in of the LH2 the was to the of units LH2 ring, which was in and for LH1 of of LH1 complex by be observed from of individual LH1 complexes from membrane of the in LH1 complexes is in polygonal rings open rings and such as arcs and were rings are in which circular or elliptical ring were The and formed the two comprising and of the of and open rings were observed at and The ring observed for LH1 of and which are to as and In the circular LH1 the of small rings was rings were and large rings were the of the we that the small rings 15 the and the large For the of and large was and The of of the LH1 complexes was by the for two For the elliptical the between and long was found to for small and large in the other that were for LH1 complexes. The of LH1 rings the lipid bilayer was which is to the of LH1 rings on the for the complex J. D. Proc. Natl. Acad. Sci. U. 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It is that this of from small differences in of these complexes at the and with a of these associations in a The flexibility of LH1 in the of in such and may the of in for in and of the LH1 and with the LH2 have forms and can no variations in either or shape for the LH2 complex. there be substantial differences between the protein of LH1 and LH2 which are apparent from a of the We that the between these complexes is the arrangement of between the of LH complexes and the of the bound R.J. McGlynn P. Jones M.R. Hunter C.N. 1998; PubMed Scopus Google a of and has that a of each within so that the to each of Bchls are by the α and β polypeptides that the same Bchls J.D. Hunter C.N. Proc. Natl. Acad. Sci. U. A. 1994; PubMed Scopus Google Scholar, J.D. Westerhuis W.H. Hunter C.N. Biochemistry. PubMed Scopus Google Scholar, Olsen J.D. Hunter C.N. Biochemistry. PubMed Scopus Google Scholar). these are to the and a significant force to stabilize this complex C.M. McGlynn P. Olsen J.D. Hunter C.N. P.A. Biochemistry. PubMed Scopus Google Scholar). In contrast, the H-bonding for the LH2 complexes from R. sphaeroides and R. acidophila each to an αβ of with the other the αβ This is in which a of the ring of the R. acidophila complex Prince S.M. Howard T. Cogdell R.J. Isaacs N.W. J. Mol. Biol. 2003; PubMed Scopus Google Scholar) in and in and of the LH1 complex in from EM, and D. Qian P. A. Bullough P.A. Engel A. Hunter C.N. J. Biol. 2004; PubMed Scopus Google and in The of these is that each within LH1 has a of within the complex. This is by units in and a of in This is by the that LH1 can be into individual P.A. Biochemistry. PubMed Scopus Google and that it can be into a series of LH1 that in from to Hunter C.N. Biochemistry. 2002; PubMed Scopus Google Scholar, Hunter C.N. van Grondelle R. J. Google Scholar). of these of of a LH2 complex has been reported, the LH2 of Rhodospirillum has been into P.A. Biochemistry. 1998; PubMed Scopus Google Scholar). provides a rationale for the R. LH2 its H-bonding arrangement that of LH1 F. H. Biochemistry. PubMed Scopus Google Scholar). In the light of these differences between LH1 and LH2 it is to LH2 displays no in either its or shape by AFM, and LH1 as a series of capable of the elliptical, and open rings in of the and of complex is to both circular and elliptical as in the (4Jamieson S.J. Wang P. Qian P. Kirkland J.Y. Conroy M.J. Hunter C.N. Bullough P.A. EMBO J. 2002; 21: 3927-3935Crossref PubMed Scopus (131) Google Scholar) and in (5Roszak A.W. Howard T.D. Southall J. Gardiner A.T. Law C.J. Isaacs N.W. Cogdell R.J. Science. 2003; 302: 1969-1972Crossref PubMed Scopus (600) Google Scholar). This implied flexibility of the LH1 complex was suggested to be for its by the export of quinol formed as a of reaction center (4Jamieson S.J. Wang P. Qian P. Kirkland J.Y. Conroy M.J. Hunter C.N. Bullough P.A. EMBO J. 2002; 21: 3927-3935Crossref PubMed Scopus (131) Google Scholar). of the complex demonstrated a which to a circular shape of the RC J. D. Proc. Natl. Acad. Sci. U. A. 2003; 100: PubMed Scopus Google Scholar). The shapes of the complex in a flexible structure was It was an ability to LH1 of or R. into circular or elliptical shapes by allow the passage of quinol from the RC to the quinone demonstration that the linkage between adjacent units is flexible and even at room temperature in a detergent-free membrane bilayer provides a rationale for the opening and of LH1 units the QB site of the The existence of open rings and arcs in to this it that LH1 can form as was by Westerhuis Hunter C.N. Biochemistry. 2002; PubMed Scopus Google Scholar) on the basis of complexes. In this is with the of the PufX polypeptide, which the of the ring of Bchls a part of the LH1 ring by with the P.A. C.M. T.G. Beatty J.T. Hunter C.N. P.A. Biochemistry. 1998; PubMed Scopus Google providing a weak of of R. sphaeroides show open at the we these adjacent to J. D. and of for of the would to an of ring the clearly show the existence of open rings as well as a of ring It be that the ring and the open are of the We that the complexes as there is no of a at either or in the that was used for the in The that are to LH1 of R. sphaeroides into the of either a mutant LH1 or the of with to of the complex P.A. PubMed Scopus Google Scholar). We that the of native in complexes the of Francia F. J. Melandri B.A. D. J. Biol. 2004; PubMed Scopus Google Scholar) used to and core complexes of R. sphaeroides, that of organization are even at this of The of the large ring by would be with enclosing an RC complex and there may be no for the of recent of reconstituted of R. sphaeroides RC-LH1-PufX complexes demonstrated the existence of a small of rings (6Siebert C.A. Qian P. Fotiadis D. Engel A. Hunter C.N. Bullough P.A. EMBO J. 2004; 23: 690-700Crossref PubMed Scopus (136) Google Scholar). The study of J. D. Proc. Natl. Acad. Sci. U. A. 2003; 100: PubMed Scopus Google Scholar) on native membranes of the have the of other ring it is the when the ring is and it this process. It is that LH1 can assemble in from its units P.A. Biochemistry. PubMed Scopus Google the of in of LH1 is it has been that there is an factor for the RCLH1 complex Beatty J.T. J. 1998; PubMed Google Scholar, Beatty J.T. J. 1998; PubMed Google Scholar). We have observed in LH1 aggregation in membranes both PufX and the RC are or both of these be an factor in the LH1 ring of R. the absence of the RC complex the of a or on which to assemble and in its in However, only in imaging of the membranes of the mutant this and this has used to demonstrate large variations from to in the LH1 light harvesting complex, in terms of both shape and size. from enclosing the RC, the inherent flexibility and lack of structural coherence of this complex In the existence of open rings and arcs provides a direct visualization of consequences of the relatively weak associations that govern the of the protomers comprising the LH1 complex. which are to be for the LH1 complex and the LH2 complex, are suggested to from the H-bonding patterns that stabilize binding of the Bchls to the LH polypeptides. This by the of the RC, forms the basis for a dynamic separation of individual protomers, passage of quinol from the RC to the quinone pool prior to reduction of the cytochrome bc1 complex. with