Exploring Masses and Internal Mass Distributions of Single Carboxysomes in Free Solution Using Fluorescence and Interferometric Scattering in an Anti-Brownian Trap

Abstract

Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by colocalizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50–400 MDa range by analyzing their scattering cross sections with a core–shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects.

Monitoring physical and chemical properties of individual carboxysomes trapped in solution

“…Reduced carboxysomes show low values of the average redox ratios R405/488. Along with the capability to estimate single-particle masses from each scattering contrast, [21] these experiments demonstrate the ability of the ISABEL trap to monitor nanoscale biological objects like carboxysomes and viruses for extended times and to expand the range of local reporter experiments that can be done in these systems.…”

Dynamic excitation for photophysical control and enhanced FRET sensing in an Anti-Brownian ELectrokinetic (ABEL) Trap

“…While single particlesas small as 1 nm [5][6][7] are trapped, they experience uniform illumination intensity and rotate freely, enabling an isotropic view of the molecule and simultaneous high-precision acquisition of multiple spectroscopic parameters including brightness, fluorescence lifetime, anisotropy, and emission spectrum. Such high information-content data have proved useful for single-molecule studies of systems which exhibit photophysical heterogeneity, such as photosynthetic antenna complexes [8][9][10][11][12][13][14] , other proteins 15,16 , aggregates and nano-compartments 17,18 , and individual nanoparticles 19,20 . ABEL trap measurements of molecules labeled with multiple dyes that undergo Förster resonance energy transfer (FRET) have demonstrated exceptional utility and future potential for generating biological insights on protein conformations 21 including enzyme motions [22][23][24][25] , protein-protein interactions 26 , and DNA structure and conformation [27][28][29][30][31] .…”

Atmospheric-Pressure Mass Spectrometry by Single-Mode Nanoelectromechanical Systems

“…Nanoelectromechanical systems (NEMS) have proven their use in the mass spectrometry field − for almost two decades, especially for analytes with masses that cannot be reached by conventional mass spectrometry techniques, i.e., >10 MDa, due to large mass-to-charge ratios ( m / z ). − Therefore, the ability to measure these high mass values allows NEMS mass spectrometry (NEMS-MS) to be a potent tool for the characterization of metallic, ceramic, polymeric, and biological nanoparticles, e.g., exosomes, viruses, and lipid vesicles. Our recent study enabled the NEMS-MS technique to work under entirely atmospheric conditions, thus opening possibilities for enhancing the NEMS-MS technique while resolving major problems such as low capture efficiencies and high system costs.…”