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Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain ofEscherichia coli(Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syntopology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26–His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

 

De novo proteins constructed from novel amino acid sequences are distinct from proteins that evolved in nature. Construct K (ConK) is a binary-patterned de novo designed protein that rescues Escherichia coli from otherwise toxic concentrations of copper. ConK was recently found to bind the cofactor PLP (pyridoxal phosphate, the active form of vitamin B 6 ). Here, we show that ConK catalyzes the desulfurization of cysteine to H 2 S, which can be used to synthesize CdS nanocrystals in solution. The CdS nanocrystals are approximately 3 nm, as measured by transmission electron microscope, with optical properties similar to those seen in chemically synthesized quantum dots. The CdS nanocrystals synthesized using ConK have slower growth rates and a different growth mechanism than those synthesized using natural biomineralization pathways. The slower growth rate yields CdS nanocrystals with two desirable properties not observed during biomineralization using natural proteins. First, CdS nanocrystals are predominantly of the zinc blende crystal phase; this is in stark contrast to natural biomineralization routes that produce a mixture of zinc blende and wurtzite phase CdS. Second, in contrast to the growth and eventual precipitation observed in natural biomineralization systems, the CdS nanocrystals produced by ConK stabilize at a final size. Future optimization of CdS nanocrystal growth using ConK—or other de novo proteins—may help to overcome the limits on nanocrystal quality typically observed from natural biomineralization by enabling the synthesis of more stable, high-quality quantum dots at room temperature. 

ABSTRACT: The dosing of peptide and protein therapeutics is complicated by rapid clearance from the blood pool and poor cellular membrane permeability. Encapsulation into nanocarriers such as liposomes or polymersomes has long been explored to overcome these limitations, but manufacturing challenges have limited clinical translation by these approaches. Recently, inverse Flash NanoPrecipitation (iFNP) has been developed to produce highly loaded polymeric nanocarriers with the peptide or protein contained within a hydrophilic core, stabilized by a hydrophobic polymer shell. Encapsulation of proteins with higher-order structure requires understanding how processing may affect their conformational state. We demonstrate a combined experimental/simulation approach to characterize protein behavior during iFNP processing steps using the Trp-cage protein TC5b as a model. Explicit-solvent fully atomistic molecular dynamics simulations with enhanced sampling techniques are coupled with two-dimensional heteronuclear multiple-quantum coherence nuclear magnetic resonance spectroscopy (2D-HMQC NMR) and circular dichroism to determine the structure of TC5b during mixed-solvent exposure encountered in iFNP processing. The simulations involve atomistic models of mixed solvents and protein to capture the complexity of the hydrogen bonding and hydrophobic interactions between water, dimethylsulfoxide (DMSO), and the protein. The combined analyses reveal structural unfolding of the protein in 11 M DMSO but confirm complete refolding after release from the polymeric nanocarrier back into an aqueous phase. 

In 2017 the Event Horizon Telescope (EHT) observed the supermassive black hole at the center of the Milky Way, Sagittarius A* (Sgr A*), at a frequency of 228.1 GHz (λ= 1.3 mm). The fundamental physics tests that even a single pulsar orbiting Sgr A* would enable motivate searching for pulsars in EHT data sets. The high observing frequency means that pulsars—which typically exhibit steep emission spectra—are expected to be very faint. However, it also negates pulse scattering, an effect that could hinder pulsar detections in the Galactic center. Additionally, magnetars or a secondary inverse Compton emission could be stronger at millimeter wavelengths than at lower frequencies. We present a search for pulsars close to Sgr A* using the data from the three most sensitive stations in the EHT 2017 campaign: the Atacama Large Millimeter/submillimeter Array, the Large Millimeter Telescope, and the IRAM 30 m Telescope. We apply three detection methods based on Fourier-domain analysis, the fast folding algorithm, and single-pulse searches targeting both pulsars and burst-like transient emission. We use the simultaneity of the observations to confirm potential candidates. No new pulsars or significant bursts were found. Being the first pulsar search ever carried out at such high radio frequencies, we detail our analysis methods and give a detailed estimation of the sensitivity of the search. We conclude that the EHT 2017 observations are only sensitive to a small fraction (≲2.2%) of the pulsars that may exist close to Sgr A*, motivating further searches for fainter pulsars in the region.

 

Event Horizon Telescope (EHT) observations have revealed a bright ring of emission around the supermassive black hole at the center of the M87 galaxy. EHT images in linear polarization have further identified a coherent spiral pattern around the black hole, produced from ordered magnetic fields threading the emitting plasma. Here we present the first analysis of circular polarization using EHT data, acquired in 2017, which can potentially provide additional insights into the magnetic fields and plasma composition near the black hole. Interferometric closure quantities provide convincing evidence for the presence of circularly polarized emission on event-horizon scales. We produce images of the circular polarization using both traditional and newly developed methods. All methods find a moderate level of resolved circular polarization across the image (〈∣v∣〉 < 3.7%), consistent with the low image-integrated circular polarization fraction measured by the Atacama Large Millimeter/submillimeter Array (∣v_int∣ < 1%). Despite this broad agreement, the methods show substantial variation in the morphology of the circularly polarized emission, indicating that our conclusions are strongly dependent on the imaging assumptions because of the limited baseline coverage, uncertain telescope gain calibration, and weakly polarized signal. We include this upper limit in an updated comparison to general relativistic magnetohydrodynamic simulation models. This analysis reinforces the previously reported preference for magnetically arrested accretion flow models. We find that most simulations naturally produce a low level of circular polarization consistent with our upper limit and that Faraday conversion is likely the dominant production mechanism for circular polarization at 230 GHz in M87*.

 

The Event Horizon Telescope (EHT) is a millimeter very long baseline interferometry (VLBI) array that has imaged the apparent shadows of the supermassive black holes M87* and Sagittarius A*. Polarimetric data from these observations contain a wealth of information on the black hole and accretion flow properties. In this work, we develop polarimetric geometric modeling methods for mm-VLBI data, focusing on approaches that fit data products with differing degrees of invariance to broad classes of calibration errors. We establish a fitting procedure using a polarimetric “m-ring” model to approximate the image structure near a black hole. By fitting this model to synthetic EHT data from general relativistic magnetohydrodynamic models, we show that the linear and circular polarization structure can be successfully approximated with relatively few model parameters. We then fit this model to EHT observations of M87* taken in 2017. In total intensity and linear polarization, the m-ring fits are consistent with previous results from imaging methods. In circular polarization, the m-ring fits indicate the presence of event-horizon-scale circular polarization structure, with a persistent dipolar asymmetry and orientation across several days. The same structure was recovered independently of observing band, used data products, and model assumptions. Despite this broad agreement, imaging methods do not produce similarly consistent results. Our circular polarization results, which imposed additional assumptions on the source structure, should thus be interpreted with some caution. Polarimetric geometric modeling provides a useful and powerful method to constrain the properties of horizon-scale polarized emission, particularly for sparse arrays like the EHT.

 

Abstract Interpretation of resolved polarized images of black holes by the Event Horizon Telescope (EHT) requires predictions of the polarized emission observable by an Earth-based instrument for a particular model of the black hole accretion system. Such predictions are generated by general relativistic radiative transfer (GRRT) codes, which integrate the equations of polarized radiative transfer in curved spacetime. A selection of ray-tracing GRRT codes used within the EHT Collaboration is evaluated for accuracy and consistency in producing a selection of test images, demonstrating that the various methods and implementations of radiative transfer calculations are highly consistent. When imaging an analytic accretion model, we find that all codes produce images similar within a pixel-wise normalized mean squared error (NMSE) of 0.012 in the worst case. When imaging a snapshot from a cell-based magnetohydrodynamic simulation, we find all test images to be similar within NMSEs of 0.02, 0.04, 0.04, and 0.12 in Stokes I , Q , U , and V , respectively. We additionally find the values of several image metrics relevant to published EHT results to be in agreement to much better precision than measurement uncertainties.