Two energy barriers and a transient intermediate state determine the unfolding and folding dynamics of cold shock protein

Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. Here we report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.

A DFT Study on the Molecular Mechanism of Additions of Electrophilic and Nucleophilic Carbenes to Non-Enolizable Cycloaliphatic Thioketones

The molecular mechanisms of addition of dihalocarbenes and dimethoxycarbene to thioketones derived from 2,2,4,4-tetrmethylcyclobutane-1,3-dione were examined on the basis of the DFT wb97xd/6-311g(d,p)(PCM) calculations. Obtained results demonstrated that the examined processes exhibit polar nature and in the case of electrophilic dichloro-, and dibromocarbenes are initiated by the attack of carbene species onto the sulfur atom of the C=S group. Remarkably, reactions involving more electrophilic carbenes (dichloro-, and dibromocarbene) proceeds via stepwise mechanism involving thiocarbonyl ylide as a transient intermediate. In contrast, analogous reactions with nucleophilic dimethoxycarbene occur via a single step reaction, which can be considered as the [2 + 1] cycloaddition reaction initiated by the attack onto the C=S bond. A computational study showed that difluorocarbene tends to react as a nucleophilic species and resembles rather dimethoxycarbene and not typical dihalocarbene species. Significantly higher reactivity of the thioketone unit in comparison to the ketone group, both present in 3-thioxo-2,2,4,4-tetramthylcyclobutanone molecule, was rationalized in the light of DFT computational study.

Free energy landscape with two barriers and a transient intermediate state determining the unfolding and folding dynamics of cold shock protein

Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. We found that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~ 8 pN, which determines a free energy landscape with two barriers and a transient intermediate between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.

Sulfoquinovose is a select nutrient of prominent bacteria and a source of hydrogen sulfide in the human gut

Responses of the microbiota to diet are highly personalized but mechanistically not well understood because many metabolic capabilities and interactions of human gut microorganisms are unknown. Here we show that sulfoquinovose (SQ), a sulfonated monosaccharide omnipresent in green vegetables, is a selective yet relevant substrate for few but ubiquitous bacteria in the human gut. In human feces and in defined co-culture, Eubacterium rectale and Bilophila wadsworthia used recently identified pathways to cooperatively catabolize SQ with 2,3-dihydroxypropane-1-sulfonate as a transient intermediate to hydrogen sulfide (H2S), a key intestinal metabolite with disparate effects on host health. SQ-degradation capability is encoded in almost half of E. rectale genomes but otherwise sparsely distributed among microbial species in the human intestine. However, re-analysis of fecal metatranscriptome datasets of four human cohorts showed that SQ degradation (mostly from E. rectale and Faecalibacterium prausnitzii) and H2S production (mostly from B. wadsworthia) pathways were expressed abundantly across various health states, demonstrating that these microbial functions are core attributes of the human gut. The discovery of green-diet-derived SQ as an exclusive microbial nutrient and an additional source of H2S in the human gut highlights the role of individual dietary compounds and organosulfur metabolism on microbial activity and has implications for precision editing of the gut microbiota by dietary and prebiotic interventions.

A precise and general FRET-based method for monitoring structural transitions in protein self-organization

Proteins assemble into a tremendous variety of dynamic and functional structures. Sensitive measurements directly in cells with a high spatiotemporal resolution are needed to distinguish these different assemblies. Here, we demonstrate precise and continuous monitoring of cytoplasmic protein self-assemblies and their structural transitions. Intermolecular FRET with both the donor and acceptor protein at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We measure different assembly structures, transient intermediate states’ kinetics, and assembly formation resolved in space and time. Specifically, the method recapitulates that i) the mutant Huntingtin exon1 (mHttex1) protein first forms low-FRET and presumably less ordered assemblies in yeast and human cells, which develop into high-FRET aggregates, ii) the chaperone DNAJB6b prevents low-FRET mHttex1 assemblies, yet coassembles with mHttex1 aggregates, and iii) FUS’ condensates have mutation-dependent nanoscopic structures. FACS measurements allow assembly measurement in a high-throughput manner crucial for screening efforts, while fluorescence microscopy provides spatiotemporally-resolved measurements on the single-condensate level during a cell’s lifetime to assess the biological consequences. Implementation in other native or non-native proteins could provide insight into many studies involving protein condensation or aggregation.

Efficient Amplification in Soai's Asymmetric Autocatalysis by a Transient Stereodynamic Catalyst

Mechanisms leading to a molecular evolution and the formation of homochirality in nature are interconnected and a key to the underlying principles that led to the emergence of life. So far proposed mechanisms leading to a non-linear reaction behavior are based mainly on the formation of homochiral and heterochiral dimers. Since homochiral and heterochiral dimers are diastereomers of each other, the minor enantiomer is shifted out of equilibrium with the major enantiomer by dimer formation and thus a reaction or catalysis can be dominated by the remaining molecules of the major enantiomer. In this article a mechanism is shown that leads to homochirality by the formation of a highly catalytically active transient intermediate in a stereodynamically controlled reaction. This is demonstrated by Soai's asymmetric autocatalysis, in which aldehydes are transformed into the corresponding alcohols by addition of dialkylzinc reagents. The mechanism of chirogenesis proposed here shows that an apparently inefficient reaction is the best prerequisite for a selection mechanism. In addition, stereodynamic control offers the advantage that the minor diastereomeric intermediate can be interconverted into the major diastereomer and thus be stereoeconomically efficient. This is supported by computer simulation of reaction kinetics.

Short-Term Foreshocks as Key Information for Mainshock Timing and Rupture: The Mw6.8 25 October 2018 Zakynthos Earthquake, Hellenic Subduction Zone

Significant seismicity anomalies preceded the 25 October 2018 mainshock (Mw = 6.8), NW Hellenic Arc: a transient intermediate-term (~2 yrs) swarm and a short-term (last 6 months) cluster with typical time-size-space foreshock patterns: activity increase, b-value drop, foreshocks move towards mainshock epicenter. The anomalies were identified with both a standard earthquake catalogue and a catalogue relocated with the Non-Linear Location (NLLoc) algorithm. Teleseismic P-waveforms inversion showed oblique-slip rupture with strike 10°, dip 24°, length ~70 km, faulting depth ~24 km, velocity 3.2 km/s, duration 18 s, slip 1.8 m within the asperity, seismic moment 2.0 × 1026 dyne*cm. The two largest imminent foreshocks (Mw = 4.1, Mw = 4.8) occurred very close to the mainshock hypocenter. The asperity bounded up-dip by the foreshocks area and at the north by the foreshocks/swarm area. The accelerated foreshocks very likely promoted slip accumulation contributing to unlocking the asperity and breaking with the mainshock. The rupture initially propagated northwards, but after 6 s stopped at the north bound and turned southwards. Most early aftershocks concentrated in the foreshocks/swarm area. This distribution was controlled not only by stress transfer from the mainshock but also by pre-existing stress. In the frame of a program for regular monitoring and near real-time identification of seismicity anomalies, foreshock patterns would be detectable at least three months prior the mainshock, thus demonstrating the significant predictive value of foreshocks.

Optimal functionalization of a molecular electrocatalyst for hydride transfer

Optimization of hydride transfer (HT) catalysts to enhance rates and selectivities of (photo)electroreduction reactions could be a crucial component of a sustainable chemical industry. Here, we analyze how ring functionalization of the adsorbed transient intermediate 2-pyridinide (2-PyH−*)—predicted to form in situ from pyridine (Py) in acidified water at a cathode surface and to be the key to selective CO2 photoelectroreduction on p-GaP—may enhance catalytic activity. Earlier studies revealed that 2-PyH−*’s instability results from a protonation side reaction producing adsorbed dihydropyridine (DHP*), which is relatively HT-inactive. Reducing the electron density on 2-PyH−* could limit this protonation, with the trade-off that it may become less active for HT from 2-PyH−*–R to CO2. We explore here how Py functionalization affects the electron distribution and in turn tunes the catalytic performance of 2-PyH−*. We indeed find that electron-withdrawing groups could enhance the stability of 2-PyH−* by reducing its electron density on the ring. Furthermore, we find that the change in the number of electrons on the substituting group of the hydride donor is a good descriptor for both the stability against protonation and the magnitude of the HT barrier. We predict that –CH2–CH2F is the best candidate substituent, as it significantly improves the stability of 2-PyH−* with only a small increase in HT barrier. –CH=CH2 and –CH2F also could be promising, although they require further investigation due to a larger HT-barrier increase.