Dataset Information

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.

SUBMITTER: Kumar S

PROVIDER: S-EPMC5269641 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Kumar Sandip S Cartron Michaël L ML Mullin Nic N Qian Pu P Leggett Graham J GJ Hunter C Neil CN Hobbs Jamie K JK

ACS nano 20161121 1

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 o ...[more]

PMID: 28114766

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Project description:We present a magnetic force-based direct drive modulation method to measure local nano-rheological properties of soft materials across a broad frequency range (10 Hz to 2 kHz) using colloid-attached atomic force microscope (AFM) probes in liquid. The direct drive method enables artefact-free measurements over several decades of excitation frequency, and avoids the need to evaluate medium-induced hydrodynamic drag effects. The method was applied to measure the local mechanical properties of polyacrylamide hydrogels. The frequency-dependent storage stiffness, loss stiffness, and loss tangent (tan??) were quantified for hydrogels having high and low crosslinking densities by measuring the amplitude and the phase response of the cantilever while the colloid was in contact with the hydrogel. The frequency bandwidth was further expanded to lower effective frequencies (0.1 Hz to 10 Hz) by obtaining force-displacement (FD) curves. Slow FD measurements showed a recoverable but highly hysteretic response, with the contact mechanical behaviour dependent on the loading direction: approach curves showed Hertzian behaviour while retraction curves fit the JKR contact mechanics model well into the adhesive regime, after which multiple detachment instabilities occurred. Using small amplitude dynamic modulation to explore faster rates, the load dependence of the storage stiffness transitioned from Hertzian to a dynamic punch-type (constant contact area) model, indicating significant influence of material dissipation coupled with adhesion. Using the appropriate contact model across the full frequency range measured, the storage moduli were found to remain nearly constant until an increase began near ?100 Hz. The softer gels' storage modulus increased from 7.9 ± 0.4 to 14.5 ± 2.1 kPa (?85%), and the stiffer gels' storage modulus increased from 16.3 ± 1.1 to 31.7 ± 5.0 kPa (?95%). This increase at high frequencies may be attributed to a contribution from solvent confinement in the hydrogel (poroelasticity). The storage moduli measured by both macro-rheometry and AFM FD curves were comparable to those measured using the modulation method at their overlapping frequencies (10-25 Hz). In all cases, care was taken to ensure the contact mechanics models were applied within the important limit of small relative deformations. This study thus highlights possible transitions in the probe-material contact mechanical behaviour for soft matter, especially when the applied strain rates and the material relaxation rates become comparable. In particular, at low frequencies, the modulus follows Hertzian contact mechanics, while at high frequencies adhesive contact is well represented by punch-like behaviour. More generally, use of the Hertz model on hydrogels at high loading rates, at high strains, or during the retraction portion of FD curves, leads to significant errors in the calculated moduli.

Dataset Information

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

Publications

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

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