Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

Abstract: The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology

Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.

Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions

Recoil-Order and Radiative Corrections to the aCORN Experiment

The aCORN experiment measures the electron-antineutrino $a$-coefficient in free neutron decay. We update the previous aCORN results to include radiative and recoil corrections to first order. The corrected combined result is $\langle a \rangle = -0.10859 \pm .00125\, ({\rm stat}) \pm 0.00133\, ({\rm sys})$, an increase in magnitude of 0.7 % compared to the overall relative standard uncertainty of 1.7 %, which is unchanged. The corresponding corrected result for the ratio of weak coupling constants $\lambda = G_A/G_V$ is $\lambda = -1.2737 \pm 0.0061$. This improves agreement with previous $a$-coefficient experiments, in particular the 2020 aSPECT result

Shape of the 8B Alpha and Neutrino Spectra

The beta-delayed alpha spectrum from the decay of 8B has been measured with a setup that minimized systematic uncertainties that affected previous measurements. Consequently the deduced neutrino spectrum presents much smaller uncertainties than the previous recommendation. The 8B neutrino spectrum is found to be harder than previously recommended with about (10-20)% more neutrinos at energies between 12-14 MeV. The efficiencies of the 37Cl, 71Ga, 40Ar, and SuperKamiokande detectors are respectively, 3.6%, 1.4%, 5.7% and 1.8% larger than previously thought.Comment: 4 pages, 5 figure

Observation of a large parity nonconserving analyzing power in Xe

A large parity nonconserving longitudinal analyzing power was discovered in polarized-neutron transmission through Xe. An analyzing power of 4.3±0.2% was observed in a p-wave resonance at En=3.2 eV. The measurement was performed with a liquid Xe target of natural isotopic abundance that was placed in the polarized epithermal neutron beam, flight path 2, at the Manuel Lujan Neutron Science Center. This apparatus was constructed by the TRIPLE Collaboration, and has been used for studies of parity symmetry in compound nuclear resonances. Part of the motivation of the experiment was to discover a nucleus appropriate for a sensitive test of time-reversal invariance in polarized-neutron transmission. The large analyzing power of the observed resonance may make it possible to design a test of time reversal invariance using a polarized-Xe target

Measurement of the neutron lifetime using an asymmetric magneto- gravitational trap and in situ detection

The precise value of the mean neutron lifetime, $\tau_n$, plays an important role in nuclear and particle physics and cosmology. It is a key input for predicting the ratio of protons to helium atoms in the primordial universe and is used to search for new physics beyond the Standard Model of particle physics. There is a 3.9 standard deviation discrepancy between $\tau_n$ measured by counting the decay rate of free neutrons in a beam (887.7 $\pm$ 2.2 s) and by counting surviving ultracold neutrons stored for different storage times in a material trap (878.5$\pm$0.8 s). The experiment described here eliminates loss mechanisms present in previous trap experiments by levitating polarized ultracold neutrons above the surface of an asymmetric storage trap using a repulsive magnetic field gradient so that the stored neutrons do not interact with material trap walls and neutrons in quasi-stable orbits rapidly exit the trap. As a result of this approach and the use of a new in situ neutron detector, the lifetime reported here (877.7 $\pm$ 0.7 (stat) +0.4/-0.2 (sys) s) is the first modern measurement of $\tau_n$ that does not require corrections larger than the quoted uncertainties.Comment: 9 pages, 3 figures, 2 table

Status of the UCNτ experiment

The neutron is the simplest nuclear system that can be used to probe the structure of the weak interaction and search for physics beyond the standard model. Measurements of neutron lifetime and β-decay correlation coefficients with precisions of 0.02% and 0.1%, respectively, would allow for stringent constraints on new physics. The UCNτ experiment uses an asymmetric magneto-gravitational UCN trap with in situ counting of surviving neutrons to measure the neutron lifetime, τ_n = 877.7s (0.7s)_(stat) (+0.4/−0.2s)_(sys). We discuss the recent result from UCNτ, the status of ongoing data collection and analysis, and the path toward a 0.25 s measurement of the neutron lifetime with UCNτ