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Careful measurements of light scattering can provide information on individual macromolecules and complexes. Young et al. used a light-scattering approach for accurate mass determination of proteins as small as 20 kDa (see the Perspective by Lee and Klenerman). Movies of protein complex association and dissociation were analyzed to extract biophysical parameters from single molecules and assemblies without labeling. Using this approach, the authors determined in vitro kinetics of fibril and aggregate growth and association constants for a complex protein-glycoprotein assembly. Science , this issue p. 423 ; see also p. 378 Light scattering allows dynamic observation of biomolecule mass, interactions, and assembly. 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.

Na + /H + antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na + /H + exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane. Na + /H + antiporters transport sodium or lithium ions across the membrane in exchange for protons. Here the authors combine ion mobility mass spectrometry and molecular dynamics simulations to uncover a facilitating role for lipids in the transport mechanism.

Transcription factors search for specific operator sequences by alternating rounds of 3D diffusion with rounds of 1D diffusion (sliding) along the DNA. The details of such sliding have largely been beyond direct experimental observation. For this purpose we devised an analytical formulation of umbrella sampling along a helical coordinate, and from extensive and fully atomistic simulations we quantified the free-energy landscapes that underlie the sliding dynamics and dissociation kinetics for the LacI dimer. The resulting potential of mean force distributions show a fine structure with an amplitude of 1 k BT for sliding and 12 k BT for dissociation. Based on the free-energy calculations the repressor slides in close contact with DNA for 8 bp on average before making a microscopic dissociation. By combining the microscopic molecular-dynamics calculations with Brownian simulation including rotational diffusion from the microscopically dissociated state we estimate a macroscopic residence time of 48 ms at the same DNA segment and an in vitro sliding distance of 240 bp. The sliding distance is in agreement with previous in vitro sliding-length estimates. The in vitro prediction for the macroscopic residence time also compares favorably to what we measure by single-molecule imaging of nonspecifically bound fluorescently labeled LacI in living cells. The investigation adds to our understanding of transcription-factor search kinetics and connects the macro-/mesoscopic rate constants to the microscopic dynamics.

Noroviruses are the major cause of viral gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. The structural aspects of glycan binding to fully deamidated GII.4 P dimers have been investigated before. However, considering the high specificity and half-life of N373D under physiological conditions, large fractions of partially deamidated virions with potentially altered dynamics in their P domains are likely to occur. Therefore, we also examined glycan binding to partially deamidated GII.4 Saga and GII.4 MI001 P dimers. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric subpopulation. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a complex role of deamidation in modulating glycan-mediated cell attachment in GII.4 strains.

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (> 90%) slides over its natural lacO₁ operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.

Most proteins assemble into multisubunit complexes 1 . The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend on multimerization, such as intersubunit allostery or the capacity to do mechanical work 2 . In many complexes, however, multimerization does not enable any known function 3 . An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function 3 – 7 . Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors, we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposure of this interface to solvent reduces protein stability and causes aggregation, even though the interface makes no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels that are not tolerated in monomers. In a database of hundreds of families of multimers, most show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous. Accumulation of hydrophobic residues at the interface between monomers may favour the maintenance of multimeric protein states during evolution, even if multimerization confers no functional advantage.

Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our findings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray diffraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.

Fractals are patterns that are self-similar across multiple length-scales 1 . Macroscopic fractals are common in nature 2 – 4 ; however, so far, molecular assembly into fractals is restricted to synthetic systems 5 – 12 . Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus , which self-assembles into Sierpiński triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution. Citrate synthase from the cyanobacterium Synechococcus elongatus is shown to self-assemble into Sierpiński triangles, a finding that opens up the possibility that other naturally occurring molecular-scale fractals exist.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm−1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.