Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy
"Direct imaging of protein organization in an intact bacterial organelle using high-resolution atomic force microscopy", S. Kumar, M. Cartron, N. Mullin, P. Qian, G. J. Leggett, C. N. Hunter and J. K. Hobbs, ACS Nano, accepted for publication.
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.
Strong Coupling of Localized Surface Plasmons to Excitons in Light-Harvesting Complexes
A. Tsargorodska, M. L. Cartron, C. Vasilev, G. Kodali, O. A. Mass, J. J. Baumberg, P. L. Dutton, C. N. Hunter, P. Törmä, and G. J. Leggett, Nano Lett. 2016, 16, 6850–6856.
Gold nanostructure arrays exhibit surface plasmon resonances that split after attaching light harvesting complexes 1 and 2 (LH1 and LH2) from purple bacteria. The splitting is attributed to strong coupling between the localized surface plasmon resonances and excitons in the light-harvesting complexes. Wild-type and mutant LH1 and LH2 from Rhodobacter sphaeroides containing different carotenoids yield different splitting energies, demonstrating that the coupling mechanism is sensitive to the electronic states in the light harvesting complexes. Plasmon–exciton coupling models reveal different coupling strengths depending on the molecular organization and the protein coverage, consistent with strong coupling. Strong coupling was also observed for self-assembling polypeptide maquettes that contain only chlorins. However, it is not observed for monolayers of bacteriochlorophyll, indicating that strong plasmon–exciton coupling is sensitive to the specific presentation of the pigment molecules.
Fabrication of Self-Cleaning, Reusable Titania Templates for Nanometer and Micrometer Scale Protein Patterning
The photocatalytic self-cleaning characteristics of titania facilitate the fabrication of reuseable templates for protein nanopatterning. Titania nanostructures were fabricated over square centimeter areas by interferometric lithography (IL) and nanoimprint lithography (NIL). For both fabrication techniques, subsequent adsorption of an oligo(ethylene glycol) functionalized trichlorosilane yielded an entirely passive, protein-resistant surface. Near-UV exposure caused removal of this protein-resistant film from the titania regions by photocatalytic degradation, leaving the passivating silane film intact on the silicon dioxide regions. Proteins labeled with fluorescent dyes were adsorbed to the titanium dioxide regions, yielding nanopatterns with bright fluorescence. Subsequent near-UV irradiation of the samples removed the protein from the titanium dioxide nanostructures by photocatalytic degradation facilitating the adsorption of a different protein. The process was repeated multiple times indicating that the samples produced using these simple processes are highly durable.
Facile Formation of Highly Mobile Supported Lipid Bilayers on Surface-Quaternized pH-Responsive Polymer Brushes
Poly(2-dimethylamino)ethyl methacrylate) (PDMA) brushes are grown from planar substrates via surface atom transfer radical polymerization (ATRP). When quaternization of these brushes is conducted using 1-iodooctadecane in n-hexane, which is a non-solvent for PDMA, reactivity is confined to the surface region, producing pH-responsive brushes that have a hydrophobic upper surface. Systematic variation of the 1-iodooctadecane concentration and reaction time enables the mean degree of surface quaternization to be optimized. However, when quaternisation is carried out in a good brush solvent (THF) cross-linking occurs throughout the brush layer and stimulus-responsiveness is lost. Surface quaternization of PDMA brushes using 1-iodooctadecane in n-hexane provides a substrate that facilitates formation of robust SLBs. Fluorescence recovery after photobleaching (FRAP) studies of such SLBs indicate diffusion coefficients (2.8 ± 0.3 μm s–1) and mobile fractions (98 ± 2%) that are comparable to the literature data reported for SLBs prepared directly on planar glass substrates.
Zwitterionic Poly(amino acid methacrylate) Brushes
A. M. Alswieleh, N. Cheng, I. Canton, B. Ustbas, X. Xue, V. Ladmiral, S. Xia, R. E. Ducker, O. El Zubir, M. L. Cartron, C. N. Hunter, G. J. Leggett and S. P. Armes, J. Am. Chem. Soc. 2014, 136, 9404-9413
A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially-available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 oC. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation. UV exposure using a photomask yielded sharp, well-defined micro-patterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nano-patterned PCysMA brushes were obtained using interference lithography and confocal microscopy again confirmed the selective conjugation of GFP. Good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH.
Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides
We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.
Fast, simple, combinatorial routes to the fabrication of reusable, plasmonically active gold nanostructures by interferometric lithography of self-assembled monolayers
Using a Lloyd’s mirror interferometer and a self-assembled monolayer of alkylthiolates as a resist, arrays of gold nanoparticles are formed over square cm areas with controllable, uniform properties that span a wide range of period, feature size and geometry. Samples are robust reusable after cleaning in piranha solution. A library of 200 different nanostructures has been prepared. Annealing yields a high degree of crystallinity and strong plasmon absorptions. Strong enhancements were observed in the Raman spectra of phthalocyanines. The shift in the position of the plasmon band after site-specific attachment of histidine-tagged green fluorescent protein (His-GFP) and bacteriochlorophyll a was measured for a range of nanostructured films, enabling the rapid identification of the one that yielded the largest shift. This approach offers a simple route to the production of durable, reusable, macroscopic arrays of gold nanostructures “to order” with preselected plasmon resonance energies.