The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental ...challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.
Optoelectronic devices based on hybrid halide perovskites have shown remarkable progress to high performance. However, despite their apparent success, there remain many open questions about their ...intrinsic properties. Single crystals are often seen as the ideal platform for understanding the limits of crystalline materials, and recent reports of rapid, high-temperature crystallization of single crystals should enable a variety of studies. Here we explore the mechanism of this crystallization and find that it is due to reversible changes in the solution where breaking up of colloids, and a change in the solvent strength, leads to supersaturation and subsequent crystallization. We use this knowledge to demonstrate a broader range of processing parameters and show that these can lead to improved crystal quality. Our findings are therefore of central importance to enable the continued advancement of perovskite optoelectronics and to the improved reproducibility through a better understanding of factors influencing and controlling crystallization.
Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery
, and have inspired synthetic biologists to create novel protein assemblies ...via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging
. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Au
-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms ...temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head-head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps.
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•Quantifying high affinity protein–protein interactions is experimentally difficult.•We use mass photometry to determine αβ-tubulin heterodimer energetics and kinetics.•The Kd of the ...dimer is 8.48 ± 1.22 nM in the absence of added GTP.•This lowers to 3.69 ± 0.65 nM upon GTP addition with a koff > 10−2 s−1.•Mass photometry is uniquely suited to study protein–protein interactions.
The αβ-tubulin heterodimer is the fundamental building block of microtubules, making it central to several cellular processes. Despite the apparent simplicity of heterodimerisation, the associated energetics and kinetics remain disputed, largely due to experimental challenges associated with quantifying affinities in the <µM range. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the αβ-tubulin dissociation constant (8.48 ± 1.22 nM) and its tightening in the presence of GTP (3.69 ± 0.65 nM), at a dissociation rate >10−2 s−1. Our results demonstrate the capabilities of mass photometry for quantifying protein–protein interactions and clarify the energetics and kinetics of tubulin heterodimerisation.
Prokaryotes have the ability to walk on surfaces using type IV pili (TFP), a motility mechanism known as twitching
. Molecular motors drive TFP extension and retraction, but whether and how these ...movements are coordinated is unknown
. Here, we reveal how the pathogen Pseudomonas aeruginosa coordinates the motorized activity of TFP to power efficient surface motility. To do this, we dynamically visualized TFP extension, attachment and retraction events at high resolution in four dimensions using label-free interferometric scattering microscopy (iSCAT)
. By measuring TFP dynamics, we found that the retraction motor PilT was sufficient to generate tension and power motility in free solution, while its partner ATPase PilU may improve retraction only in high-friction environments. Using precise timing of successive attachment and retraction, we show that P. aeruginosa engages PilT motors very rapidly and almost only when TFP encounter the surface, suggesting contact sensing. Finally, measurements of TFP dwell times on surfaces show that tension reinforced the adhesion strength to the surface of individual pili, thereby increasing effective pulling time during retraction. The successive control of TFP extension, attachment, retraction and detachment suggests that sequential control of motility machinery is a conserved strategy for optimized locomotion across domains of life.
A review and analysis of recent attempts to promote local economic development and improve public services that considers the potential role of the ‘growth coalitions’ and ‘smart local government’ ...project in progressing and creating synergy between these two agendas. A critique of the current growth agenda, as represented by Local Economic Partnerships and the Single Local Growth Fund and whether it connects with the potential growth opportunities that can be realised out of local public service expenditure. A consideration of whether Community Budgets, as the current approach to improving local public services, represent a new managerialism rather than a new ‘transformation’ agenda to better meet the needs and address the well-being of local people. Suggestions are made about how to capture the inherent value, and realise transformational change in the provision of local public service services along the lines of the decade-old ‘growth coalitions’ and ‘smart local government’ project. Also, about adding value by creating synergy between local public service expenditure and the work of Local Economic Partnerships and the Single Local Growth Fund, for the benefit of local citizens and growth.
The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies ...local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.
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