Characterization of membrane protein interactions by peptidisc-mediated mass photometry.

Membrane proteins perform numerous critical functions in the cell, making many of them primary drug targets. However, their preference for a lipid environment makes them challenging to study using established solution-based methods. Here, we show that peptidiscs, a recently developed membrane mimetic, provide an ideal platform to study membrane proteins and their interactions with mass photometry (MP) in detergent-free conditions. The mass resolution for membrane protein complexes is similar to that achievable with soluble proteins owing to the low carrier heterogeneity. Using the ABC transporter BtuCD, we show that MP can quantify interactions between peptidisc-reconstituted membrane protein receptors and their soluble protein binding partners. Using the BAM complex, we further show that MP reveals interactions between a membrane protein receptor and a bactericidal antibody. Our results highlight the utility of peptidiscs for membrane protein characterization in detergent-free solution and provide a rapid and powerful platform for quantifying membrane protein interactions.

Roadmap on Label-Free Super-Resolution Imaging.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

Surface-Enhanced Raman Scattering and Surface-Enhanced Infrared Absorption by Plasmon Polaritons in Three-Dimensional Nanoparticle Supercrystals.

Surface-enhanced vibrational spectroscopy strongly increases the cross section of Raman scattering and infrared absorption, overcoming the limited sensitivity and resolution of these two powerful analytic tools. While surface-enhanced setups with maximum enhancement have been studied widely in recent years, substrates with reproducible, uniform enhancement have received less attention although they are required in many applications. Here, we show that plasmonic supercrystals are an excellent platform for enhanced spectroscopy because they possess a high density of hotspots in the electric field. We describe the near field inside the supercrystal within the framework of plasmon polaritons that form due to strong light-matter interaction. From the polariton resonances we predict resonances in the far-field enhancement for Raman scattering and infrared absorption. We verify our predictions by measuring the vibrations of polystyrene molecules embedded in supercrystals of gold nanoparticles. The intensity of surface-enhanced Raman scattering is uniform within 10% across the crystal with a peak integrated enhancement of up to 300 and a peak hotspot enhancement of 105. The supercrystal polaritons induce pairs of incoming and outgoing resonances in the enhanced cross section as we demonstrate experimentally by measuring surface-enhanced Raman scattering with multiple laser wavelengths across the polariton resonance. The infrared absorption of polystyrene is likewise enhanced inside the supercrystals with a maximum enhancement of 400%. We show with a coupled oscillator model that the increase originates from the combined effects of hotspot formation and the excitation of standing polariton waves. Our work clearly relates the structural and optical properties of plasmonic supercrystals and shows that such crystals are excellent hosts and substrates for the uniform and predictable enhancement of vibrational spectra.

Infrared Scattering-Type Scanning Near-Field Optical Microscopy of Biomembranes in Water.

Infrared (IR) absorption spectroscopy detects the state and chemical composition of biomolecules solely by their inherent vibrational fingerprints. Major disadvantages like the lack of spatial resolution and sensitivity have lately been overcome by the use of pointed probes as local sensors enabling the detection of quantities as few as hundreds of proteins with nanometer precision. However, the strong absorption of infrared radiation by liquid water still prevents simple access to the measured quantity: the light scattered at the probing atomic force microscope tip. Here we report on the local IR response of biological membranes immersed in aqueous bulk solution. We make use of a silicon solid immersion lens as the substrate and focusing optics to achieve detection efficiencies sufficient to yield IR near-field maps of purple membranes. Finally, we suggest a means to improve the imaging quality by tracing the tip by a laser-scanning approach.

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase.

Hydrogenases are among the fastest H2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species.

Disc Antenna Enhanced Infrared Spectroscopy: From Self-Assembled Monolayers to Membrane Proteins.

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 104 giving rise to a detection limit <1 femtomol (10-15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region.

Rigid DNA beams for high-resolution single-molecule mechanics.

Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.