pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.

Mapping the electrostatic potential of the nucleosome acidic patch

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. We present a refined solution structure of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. We show that the conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and that most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89 we find strong evidence for an elevated pKa of 5.3. Our data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.

Deprotometalation-Iodolysis and Direct Iodination of 1-Arylated 7-Azaindoles: Reactivity Studies and Molecule Properties

Five protocols were first compared for the copper-catalyzed C-N bond formation between 7-azaindole and aryl/heteroaryl iodides/bromides. The 1-arylated 7-azaindoles thus obtained were subjected to deprotometalation-iodolysis sequences using lithium 2,2,6,6-tetramethylpiperidide as the base and the corresponding zinc diamide as an in situ trap. The reactivity of the substrate was discussed in light of the calculated atomic charges and the pKa values. The behavior of the 1-arylated 7-azaindoles in direct iodination was then studied, and the results explained by considering the HOMO orbital coefficients and the atomic charges. Finally, some of the iodides generated, generally original, were involved in the N-arylation of indole. While crystallographic data were collected for fifteen of the synthesized compounds, biological properties (antimicrobial, antifungal and antioxidant activity) were evaluated for others.

Quantifying charge state heterogeneity for proteins with multiple ionizable residues

For proteins with multiple ionizable residues, the canonical assumption is that ionization states of residues are fixed by their intrinsic pKa values. However, several studies have shown that protonation / deprotonation of acidic vs. basic sidechains is realizable even when the solution pH is kept fixed at values that are far away from the intrinsic pKa values. Indeed, protein solutions are best described as ensembles of charge microstates, with each member of the ensemble being a distinct charge microstate defined by differences in charge states for ionizable residues. Accordingly, for a given set of solution conditions, the true partition function is sum over all charge microstates and all the Boltzmann weights of all conformations associated with each of the charge microstates. Here, we leverage the advantages afforded by potentiometric titrations to measure global net charge as a function of pH, independent of considerations of conformational preferences. The systems studied are fragments of proteins with repetitive patterns of Lys and Glu. We analyze the potentiometry data using the recently introduced formalism of the q-canonical ensemble. In this ensemble, charge microstates can be grouped into mesostates. Each mesostate is a collection of microstates of the same net charge. We analyze data for global charge vs. pH to extract mesostate populations as a function of pH. Our findings reveal that the heterogeneity of charge states makes significant contributions to measured charge profiles. This has significant implications for the types of species that are present in solution, even for a fixed pH. Measurements of net charge, decoupled from measurements of conformational equilibria, and analyzed to extract the pH-dependent populations of different mesostates, will be significant for accurate understanding of how charge state heterogeneity contributes to conformational, binding, and phase equilibria of proteins, especially those that are intrinsically disordered.

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria

Ratiometric near-infrared fluorescent probes (AH+ and BH+) have been prepared for pH determination in mitochondria by attaching dithioacetal and formal residues onto a hemicyanine dye. The reactive formyl group on probe BH+ allows for retention inside mitochondria as it can react with a protein primary amine residue to form an imine under slightly basic pH 8.0. Probes AH+ and BH+ display ratiometric fluorescent responses to pH changes through the protonation and deprotonaton of a hydroxy group in hemicyanine dyes with experimentally determined pKa values of 6.85 and 6.49, respectively. Calculated pKa values from a variety of theoretical methods indicated that the SMDBONDI method of accounting for solvent and van der Waals radii plus including a water molecule located near the site of protonation produced the closest overall agreement with the experimental values at 7.33 and 6.14 for AH+ and BH+ respectively.

Comparison of various organic acids for xylo-oligosaccharide productions in terms of pKa values and combined severity

Background Methods to produce XOS have been intensively investigated, including enzymatic hydrolysis, steam explosion, and acid hydrolysis. Acid hydrolysis is currently the most widely used method to produce XOS due to its advantages of fewer processing steps, stronger raw material adaptability, higher yield, and better reproducibility. Especially, organic acids such as acetic acid, formic acid and xylonic acid work better as compared with mineral acids. However, the catalytic mechanism of different organic acids has been little studied. In this paper, four different organic acids, including formic acid, glycolic acid, lactic acid, and acetic acid were selected to compare their hydrolytic effects. Results Using pKa values as the benchmark, the yield of xylo-oligosaccharide (XOS) increased with the increasing value of pKa. The yield of XOS was 37% when hydrolyzed by 5% acetic acid (pKa = 4.75) at 170 ℃ for 20 min. Combined severity (CS), a parameter associated with temperature and reaction time was proposed, was proposed to evaluate the hydrolysis effect. The results of CS were consistent with that of pKa values on both the yield of XOS and the inhibitor. Conclusion The results based on pKa values and combined severity, a parameter associated with temperature and reaction time, concluded that acetic acid is a preferred catalyst. Combining the techno-economic analysis and environmental benefits, acetic acid hydrolysis process has lower factory production costs, and it is also an important metabolite and a carbon source for wastewater anaerobic biological treatment. In conclusion, production of xylo-oligosaccharides by acetic acid is an inexpensive, environment-friendly, and sustainable processing technique.

Comparison of various orgainc acids for xylo-oligosaccharide productions in term of pKa values and combined severity

Background: Methods to produce XOS have been intensively investigated, including enzymatic hydrolysis, steam explosion, and acid hydrolysis. Acid hydrolysis is currently the most widely used method to produce XOS due to its advantages of fewer processing steps, stronger raw material adaptability, higher yield, and better reproducibility. Especially, organic acids such as acetic acid, formic acid and xylonic acid work better as compared with mineral acids. However, the catalytic mechanism of different organic acids has been little studied. In this paper, four different organic acids, including formic acid, glycolic acid, lactic acid, and acetic acid were selected to compare their hydrolytic effects.Results: Using pKa values as the benchmark, the yield of xylo-oligosaccharide (XOS) increased with the increasing value of pKa. The yield of XOS was 37% when hydrolyzed by 5% acetic acid (pKa=4.75) at 170℃ for 20 min. Combined severity (CS), a parameter associated with temperature and reaction time was proposed, was proposed to evaluate the hydrolysis effect. The results of CS were consistent with that of pKa values on both the yield of XOS and the inhibitor.Conclusion: The results based on pKa values and combined severity, a parameter associated with temperature and reaction time, concluded that acetic acid is a preferred catalyst. Combining the techno-economic analysis and environmental benefits, acetic acid hydrolysis process has lower factory production costs, and it is also an important metabolite and a carbon source for wastewater anaerobic biological treatment. In conclusion, production of xylo-oligosaccharides by acetic acid is an inexpensive, environment-friendly, and sustainable processing technique.