Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

Kumar, Sandip; Cartron, Michaël L.; Mullin, Nic; Qian, Pu; Leggett, Graham J.; Hunter, C. Neil; Hobbs, Jamie K.

doi:10.1021/acsnano.6b05647

Title: Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

Journal Article · Mon Nov 14 00:00:00 EST 2016 · ACS Nano

DOI:https://doi.org/10.1021/acsnano.6b05647· OSTI ID:1333026

Kumar, Sandip ^[1]; Cartron, Michaël L. ^[1]; Mullin, Nic ^[1]; Qian, Pu ^[1];

^[1];

^[1]

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.

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.

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Research Organization:: Energy Frontier Research Centers (EFRC) (United States). Photosynthetic Antenna Research Center (PARC)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); Engineering and Physical Sciences Research Council (EPSRC); Biotechnology and Biological Sciences Research Council (BBSRC) (United Kingdom); European Research Council (ERC)

Grant/Contract Number:: SC 0001035; SC0001035; EP/I012060/1; BB/L014904/1; EP/M027430/1; BB/M00026/1; 338895

OSTI ID:: 1333026

Alternate ID(s):: OSTI ID: 1388745

Journal Information:: ACS Nano, Journal Name: ACS Nano Vol. 11 Journal Issue: 1; ISSN 1936-0851

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 33 works

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59 BASIC BIOLOGICAL SCIENCES
Rhodobacter sphaeroides
chromatophores
high-speed AFM
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light-harvesting 2 (LH2)
RC-LH1-PufX
ATP-synthase (ATPase)
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Title: Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

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