title: Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy.
abstract: The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip-sample interaction in both lateral and vertical directions. Here, long-range tip-sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic "organelles", chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles.
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: Sandip
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: Michaël L
authors.initials: ML
authors.lastname: Cartron
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: Nic
authors.initials: N
authors.lastname: Mullin
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: Pu
authors.initials: P
authors.lastname: Qian
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: Graham J
authors.identifier: 0000-0002-4315-9076
authors.initials: GJ
authors.lastname: Leggett
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: C Neil
authors.identifier: 0000-0003-2533-9783
authors.initials: CN
authors.lastname: Hunter
authors.affiliation: Department of Physics and Astronomy, ‡Department of Molecular Biology and Biotechnology, §Department of Chemistry, and ∥Krebs Institute, University of Sheffield , Sheffield, South Yorkshire S10 2TN, U.K.
authors.forename: Jamie K
authors.identifier: 0000-0002-5872-1404
authors.initials: JK
authors.lastname: Hobbs
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D015388:Organelles / Q000737:chemistry* / Q000378:metabolism; D010316:Particle Size; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins
keywords: ATP-synthase (ATPase); RC-LH1-PufX; Rhodobacter sphaeroides; chromatophores; high-speed AFM; light-harvesting 2 (LH2); native curved membranes