Neutral Residues Research Articles

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B-NS3 protease.

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobicd-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors.

Interaction of Statherin-Derived Peptide with the Surface of Hydroxyapatite: Perspectives Based on Molecular Dynamics Simulations

Introduction: Statherin-derived peptide (StatpSpS) has shown promise against erosive tooth wear. To elucidate its interaction with the hydroxyapatite (HAP) surface, the mechanism related to adsorption of this peptide with HAP was investigated through nanosecond-long all-atom molecular dynamics simulations. Methods: StatpSpS was positioned parallel to the HAP surface in 2 orientations: 1 – neutral and negative residues facing the surface and 2 – positive residues facing the surface. A system containing StatpSpS without HAP was also simulated as control. In the case of systems with HAP, both partially restrained surface and unrestrained surface were constructed. Structural analysis, interaction pattern, and binding-free energy were calculated. Results: In the peptide system without the HAP, there were some conformational changes during the simulation. In the presence of the surface, only moderate changes were observed. Many residues exhibited short and stable distances to the surface, indicating strong interaction. Specially, the residues ASP1 and SER2 have an important role to anchor the peptide to the surface, with positively charged residues, mainly arginine, playing a major role in the further stabilization of the peptide in an extended conformation, with close contacts to the HAP surface. Conclusion: The interaction between StatpSpS and HAP is strong, and the negative charged residues are important to the anchoring of the peptide in the surface, but after the initial placement the peptide rearranges itself to maximize the interactions between positive charged residues.

Gold nanoparticle-mediated molecularly imprinted electrochemical sensor MIP/AuNPs/GCE for highly sensitive and selective detection of neutral phosmet residues in fruits and vegetables

A novel electrochemical sensor MIP/AuNPs/GCE was developed by depositing gold nanoparticles and electropolymerizing molecularly imprinted film on the surface of glassy carbon electrode (GCE) for highly sensitive detection of neutral phosmet residues in fruits and vegetables. Scanning electron microscopy, energy dispersive spectroscopy, atomic force microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the imprinted sensor. The results showed that the imprinted film on GCE was evenly prepared with better specific recognition of phosmet on MIP/AuNPs/GCE than that on bare GCE. Several parameters including deposition time, functional monomer and its molar ratio, scanning cycles, and elution time were optimized in detail. The linear range of the MIP/AuNPs/GCE prepared in this work was 0.020–4.0 nmol/L with limit of detection of 0.010 nmol/L, which was much lower than MIP/GCE (0.17 nmol/L). The identification mechanism of MIP/AuNPs/GCE for phosmet was also confirmed as adsorption-diffusion model with effective surface area increasing after mediated by AuNPs. Selectivity, reproducibility and repeatability of the MIP/AuNPs/GCE were acceptable. The recovery of applying the sensor to detect PM in real samples ranged from 91.40 % to 107.3 %, which agreed well with those of HPLC. The method of developing MIP/AuNPs/GCE sensor herein could be extended to detecting other neutral pesticide residues in food and environment.

Sequence-based prediction of the effects of histones H1 post-translational modifications: impact on the features related to the function

Post-translational modifications modulate histones H1 activity but their impact on proteins features was not studied so far. Therefore, this work was intended to answer how the most common modifications, i.e. acetylation, methylation, phosphorylation and ubiquitination, can influence on histones H1 to alter their physicochemical and molecular properties. Investigations were done with the use of sequence-based predictors trained on various protein features. Because a full set of histones H1 modifications is not included in the databases of histone proteins, the survey was performed on the human, animals, plants, fungi and protist sequences selected from UniProtKB/Swiss-Prot database. Quantitative proportions of modifications were similar between the groups of organisms (CV = 0.11) but different within the group (p < 0.05). The effects of modifications were evaluated with the use of mutated sequences obtained through the substitution of modified residue of Lys, Ser and Thr by a neutral residue of the Ala. An advantage of deleterious mutations at the sites of acetylation, methylation and ubiquitination over the sites of phosphorylation (p < 0.05) indicate that this modification have more redundant character. Modifications evoke an increase of protein solubility and stability as well as acceleration of folding kinetics and a weaken of binding affinity. Besides, they also maintain a higher extent of intrinsic structural disorder. The obtained results prove that modifications should be perceived as relevant factors influencing physicochemical features determining molecular properties. Thus, histones H1 functioning is strictly correlated with the status of modifications. Communicated by Ramaswamy H. Sarma

Tryptophan to Tryptophan Hole Hopping in an Azurin Construct.

Electron transfer (ET) between neutral and cationic tryptophan residues in the azurin construct [ReI(H126)(CO)3(dmp)](W124)(W122)CuI (dmp = 4,7-Me2-1,10-phenanthroline) was investigated by Born-Oppenheimer quantum-mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) simulations. We focused on W124•+ ← W122 ET, which is the middle step of the photochemical hole-hopping process *ReII(CO)3(dmp•-) ← W124 ← W122 ← CuI, where sequential hopping amounts to nearly 10,000-fold acceleration over single-step tunneling (ACS Cent. Sci. 2019, 5, 192-200). In accordance with experiments, UKS-DFT QM/MM/MD simulations identified forward and reverse steps of W124•+ ↔ W122 ET equilibrium, as well as back ET ReI(CO)3(dmp•-) → W124•+ that restores *ReII(CO)3(dmp•-). Strong electronic coupling between the two indoles (≥40 meV in the crossing region) makes the productive W124•+ ← W122 ET adiabatic. Energies of the two redox states are driven to degeneracy by fluctuations of the electrostatic potential at the two indoles, mainly caused by water solvation, with contributions from the protein dynamics in the W122 vicinity. ET probability depends on the orientation of Re(CO)3(dmp) relative to W124 and its rotation diminishes the hopping yield. Comparison with hole hopping in natural systems reveals structural and dynamics factors that are important for designing efficient hole-hopping processes.

Effects of Hydrogen Peroxide on the Hydrogen Bonding Structure and Dynamics of Water and Its Influence on the Aqueous Solvation of the Insulin Monomer.

The hydrogen bond structure and dynamics of water and hydrogen peroxide (H2O2) in their binary mixtures have been studied at 298 K by classical molecular dynamics simulations. Twelve different concentrations of aqueous-H2O2 solutions are considered for this study. We have analyzed the interactions between water and H2O2 by site-site pair correlation functions and observed that the probability of formation of OW···HP hydrogen bonds are higher compared to OP···HW. The second solvation shell of water is strongly affected by increasing H2O2 concentrations (XP > 0.50), which signifies the destruction of the tetrahedral network structure of water. The translational and rotational dynamics of water and H2O2 do not significantly change up to 25% of H2O2 in aqueous mixtures. The hydrogen bond lifetime of water-water, water-H2O2, and H2O2-H2O2 in the aqueous-H2O2 solutions shows a very minimal change with increasing H2O2 concentrations. In addition to this, we also investigated the effect of H2O2 on the insulin monomer and observed that higher concentrations of H2O2 (XP = 0.10) change the secondary structure. The influence of H2O2 is more on chain-B than that on chain-A in the insulin monomer. The H2O2 occupancy at the protein surface is higher for negatively charged (GLU) and polar (ASN and THR) amino acid residues compared with that for positively charged and neutral residues in the solutions.

Improvement of the fibrinolytic activity, acid resistance and thermostability of nattokinase by surface charge engineering

Nattokinase is a promising thrombolytic drug due to its powerful fibrinolytic effect and few side effects. However, the low fibrinolytic activity and stability of nattokinase have limited its industrial production and oral application. In this study, the basic and neutral amino acid residues on the surface of recombinant nattokinase AprY from Bacillus mojavensis LY-06 (rAprY) were mutated to acidic amino acid residues by surface charge engineering strategy, and two variants K12D and N109D with 92.6 % and 8.4 % increased fibrinolytic activity were obtained. The R45E variant with enhanced acid stability and thermostability was also screened, its acid stability at pH 4 and t1/2 at 55 °C were 3.7-fold and 1.8-fold higher than that of wild type rAprY, respectively. Bioinformatics analysis showed that the increased activities of K12D and N109D variants were related to the increased flexibility of the region around their active centers. The increased rigidity of 97–103 amino acid residues around the active center of R45E may be the reason for its enhanced stability and reduced catalytic activity. The multipoint mutation K12D-N109D (M2)’s catalytic activity did not increase cumulatively, but its pH stability did. The nattokinase variants generated in this study have potential for industrial production and application.

The Nβ motif of NaTrxh directs secretion as an endoplasmic reticulum transit peptide and variations might result in different cellular targeting.

Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by the signal recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nβ, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues in plant proteins-a negative residue in position 8 is suggested in animal proteins-to maintain the Nβ motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nβ motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

ObjectiveEnhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. MethodNAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). ResultNAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. ConclusionNAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

Electrostatics-Based Computational Design and Experimental Analysis of Buforin II Antimicrobial Peptide Variants with Increased DNA Affinities.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics in the treatment of bacterial infections in part due to their targeting of generic bacterial structures that make it more difficult to develop drug resistance. In this study, we introduce and implement a design workflow to develop more potent AMPs by improving their electrostatic interactions with DNA, which is a putative intracellular target. Using the existing membrane-translocating AMP buforin II (BF2) as a starting point, we use a computational workflow that integrates electrostatic charge optimization, continuum electrostatics, and molecular dynamics simulations to suggest peptide positions at which a neutral BF2 residue could be substituted with arginine to increase DNA-binding affinity either significantly or minimally, with the latter choice done to determine whether AMP binding affinity depends on charge distribution and not just overall monopole. Our analyses predicted that T1R and L8R BF2 variants would yield substantial and minimal increases in DNA-binding affinity, respectively. These predictions were validated with experimental peptide-DNA binding assays with additional computational analyses providing structural insights. Additionally, experimental measurements of antimicrobial potency showed that a design to increase DNA binding can also yield greater potency. As a whole, this study takes initial steps to support the idea that (i) a design strategy aimed to increase AMP binding affinity to DNA by focusing only on electrostatic interactions can improve AMP potency and (ii) the effect on DNA binding of increasing the overall peptide monopole via arginine substitution depends on the position of the substitution. More broadly, this design strategy is a novel way to increase the potency of other membrane-translocating AMPs that target nucleic acids.

Structural changes of polysaccharides from Astragulus after honey processing and their bioactivities on human gut microbiota.

Astragali Radix (also known as Astragulus) is a traditional medicinal and edible homologous plant for tonifying Qi. Honey-processed Astragalus is a dosage form of Astragali Radix processed with honey, which exhibited better efficacy of tonifying Qi than the raw product. Polysaccharides are their main active components. APS2a and HAPS2a were initially isolated from Astragulus and honey-processed Astragulus. Both of them are highly branched acidic heteropolysaccharides containing ɑ-configuration and β-configuration glycosidic bonds. The molecular weight and the molecular dimension of HAPS2a decreased and the GalA contained in APS2a was converted to Gal in HAPS2a. The α-configuration galactose residue 1,3,4-α-Galp in the backbone of APS2a was converted to the corresponding β-configuration galactose residue 1,3,4-β-Galp in the backbone of HAPS2a and the uronic acid residue T-α-GalpA in the sidechain of APS2a was converted to the corresponding neutral residue T-α-Galp in the side chain of HAPS2a. Bioactivity results showed that HAPS2a had better probiotic effects on Bacteroides ovatus, Bacteroides thetaiotaomicron, Bifidobacterium longum and Lactobacillus rhamnosus strains than APS2a. After degradation, the molecular weights of HAPS2a and APS2a decreased with the changes in their monosaccharide composition. The contents of total short-chain fatty acids (SCFAs) and other organic acids in HAPS2a group were higher than APS2a group. Two novel high-molecular-weight polysaccharides named APS2a and HAPS2a had different probiotic activities in vitro, which might be due to their structural differences before and after honey processing. Both of them might be possibly used as an immunopotentiator in healthy foods or dietary supplement. © 2023 Society of Chemical Industry.

An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX.

Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non-conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro-environment of the isoalloxazine ring of FAD for the substrate-FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non-conserved residues can form a conserved element to maintain the function of CPO or PPO.

Computational aided design of a halotolerant CMP kinase for enzymatic synthesis of cytidine triphosphate.

The current biocatalytic method of industrial Cytidine triphosphate (CTP) production suffers from reaction rate loss. It is caused by gradually increasing acetate salt concentration, which inhibits enzyme activities and decreases the final yield. This work gave a possible solution to this problem through computational aided design of CMP kinase (CMPK), an enzyme in the CTP production system, to increase its stability in solution with high acetate salt concentration. Enlightened by the features of natural halophilic enzymes, the basic and neutral surface residues were replaced with acidic amino acids. This protein design strategy effectively increased the activity of CMPK in the working condition (acetate concentration over 1200mM). The halotolerant CMPK was applied in fed-batch production of CTP. The maximum titer was 201.4 ± 1.6mM, and the productivity was 12.6mML-1h-1, increased 26.4% and 27.8% from the process using wild-type CMPK, respectively.

Rational design of a novel halotolerant ATP regeneration system for biocatalytic CTP production

AbstractBACKGROUNDAcetyl phosphate/acetate kinase system is a cost‐saving and efficient ATP regeneration system. However, it suffers from accumulation of salts during catalytic reactions. It inhibits the activity of acetate kinase (ACK), a crucial enzyme of this process, and hence decreases the rate of product accumulation and yield of CTP.RESULTSA new halotolerant ACK enzyme based ATP regeneration platform was established in this research by applying the concept of rational design to optimize its building blocks. Selection of residues for rational design followed several rules. First, the less conserved residue positions on the protein surface were considered. Second, avoiding significant residue interactions, suchas salt bridges. Third, following the feature of nature occurring halotolerant enzymes, the basic and neutral surface residues were replaced with acidic ones. Our mutants represent high catalytic activities and high IC50 value (inhibit activity by 50%) at high salt concentrations. Also, when the halotolerant ACK was applied to catalyze CTP production, the maximum titer was 213.5 ± 1.6 mM (which is 12.3‐fold higher than that of the traditional process), the productivity was 13.3 ± 0.1 mM L−1 h−1, and the molar yield to 5′‐cytidylate monophosphate (CMP) and utilization efficiency of energy were 92.4% and 72.7%, respectively.CONCLUSIONThis strategy can be applied to modify other enzymes in the industry and gives insight into resolving salt accumulation during biocatalytic processes. In addition, the halotolerant Adenosine Triphosphate (ATP) regeneration system built in this work can be applied to the synthesis of several other high energy compounds. © 2023 Society of Chemical Industry (SCI).

Human monocarboxylate transporters accept and relay protons via the bound substrate for selectivity and activity at physiological pH.

Human monocarboxylate/H+ transporters, MCT, facilitate the transmembrane translocation of vital weak acid metabolites, mainly l-lactate. Tumors exhibiting a Warburg effect rely on MCT activity for l-lactate release. Recently, high-resolution MCT structures revealed binding sites for anticancer drug candidates and the substrate. Three charged residues, Lys 38, Asp 309, and Arg 313 (MCT1 numbering) are essential for substrate binding and initiation of the alternating access conformational change. However, the mechanism by which the proton cosubstrate binds and traverses MCTs remained elusive. Here, we report that substitution of Lys 38 by neutral residues maintained MCT functionality in principle, yet required strongly acidic pH conditions for wildtype-like transport velocity. We determined pH-dependent biophysical transport properties, Michaelis-Menten kinetics, and heavy water effects for MCT1 wildtype and Lys 38 mutants. Our experimental data provide evidence for the bound substrate itself to accept and shuttle a proton from Lys 38 to Asp 309 initiating transport. We have shown before that substrate protonation is a pivotal step in the mechanisms of other MCT-unrelated weak acid translocating proteins. In connection with this study, we conclude that utilization of the proton binding and transfer capabilities of the transporter-bound substrate is probably a universal theme for weak acid anion/H+ cotransport.

Sequence variety in the CC’ loop of Siglec-8/9/3 determines the recognitions to sulfated oligosaccharides

Siglecs are important lectins found in different types of immune cells and function as regulatory molecules by recognizing self-associated glycans and converting extracellular interactions into signals for inhibiting immune cell functions. Although many Siglecs have been found to show broad specificities and recognize different types of sulfated oligosaccharides, Siglec-8 and Siglec-9 displayed a high degree of specificity for sialyl N-acetyllactosamine (sLacNAc) with sulfations at O6-positions of the galactose (6’-sulfation) and N-acetylglucosamine (6-sulfation), respectively. Siglec-3 was recently discovered to bind sLacNAc both sulfations. In addition to a conserved arginine residue for binding to sialic acid residue, the sequence variety in the CC’ loop may provide binding specificities to sulfated oligosaccharides in Siglecs. Thus, the present study employed molecular models to study the impact of different residues in the CC’ loops of Siglec-8/9/3 to the recognitions of 6-sulfations in Gal and/or GlcNAc of sLacNAc. The negatively charged residues in the CC’ loop of Siglec-9 formed unfavorable electrostatic repulsions with the 6-sulfate in Gal and resulted no recognitions, in contrast to the favorable interactions formed between the positively charged residues in the CC’ loop of Siglec-8 and the 6-sulfate in Gal resulting strong specificity. A two-state binding model was proposed for Siglec-3 recognizing 6-sulfations in Gal and GlcNAc of sLacNAc, as the neutral residues in the CC’ loop of Siglec-3 could not form strong favorable interactions to lock the 6-sulfate in Gal within a single binding pose or strong unfavorable interactions to repel the 6-sulfate in Gal. The oligosaccharide adopted two distinctive binding poses and oriented the sulfate groups to form interactions with residues in the CC’ loop and G-strand. The present study provided a structural mechanism for the sequence variety in the CC’ loop of Siglec-8/9/3 determining the recognitions to the sulfated oligosaccharides and offered insights into the binding specificities for Siglecs.

Computing the Differences between Asn-X and Gln-X Deamidation and Their Impact on Pharmaceutical and Physiological Proteins: A Theoretical Investigation Using Model Dipeptides

Protein deamidation is a degradation mechanism that significantly impacts both pharmaceutical and physiological proteins. Deamidation impacts two amino acids, Asn and Gln, where the net neutral residues are converted into their acidic forms. While there are multiple similarities between the reaction mechanisms of the two residues, the impact of Gln deamidation has been noted to be most significant on physiological proteins while Asn deamidation has been linked to both pharmaceutical and physiological proteins. For this purpose, we sought to analyze the thermochemical and kinetic properties of the different reactions of Gln deamidation relative to Asn deamidation. In this study, we mapped the deamidation of Gln-X dipeptides into Glu-X dipeptides using density functional theory (DFT). Full network mapping facilitated the prediction of reaction selectivity between the two primary pathways, as well as between the two products of Gln-X deamidation as a function of solvent dielectric. To achieve this analysis, we studied a total of 77 dipeptide reactions per solvent dielectric (308 total reactions). Modeled at a neutral pH and using quantum chemical and statistical thermodynamic methods, we computed the following values: enthalpy of reaction (ΔHRXN), entropy (ΔSRXN), Gibbs free energy of reaction (ΔGRXN), activation energy (EA), and the Arrhenius preexponential factor (log(A)) for each dipeptide. Additionally, using chemical reaction principles, we generated a database of computed rate coefficients for all possible N-terminus Gln-X deamidation reactions at a neutral pH, predicted the most likely deamidation reaction mechanism for each dipeptide reaction, analyzed our results against our prior study on Asn-X deamidation, and matched our results against qualitative trends previously noted by experimental literature.

Salty Enzymes: Protein Splicing Dependence on Salt Concentrations

Halophiles are organisms that thrive in very high salt concentrations. Enzymes from halophiles tend to have a negative surface charge and a low isoelectric point to allow for stable folding. We are interested in converting a halophilic enzyme that requires high salt for activity and proper folding into one that is functional at more moderate salt concentrations. Our model enzyme is DNA polymerase II from Halobacterium salinarum, which contains an intein. Inteins are intervening peptide sequences within proteins that are spliced out from their flanking polypeptides, or exteins, which then ligate to form a functional protein. We have induced mutations to the intein that facilitate a splicing reaction at lower salt concentrations. Changing negatively charged amino acids on the surface of the intein to corresponding neutral or positive charged residues observed in homologous inteins in non‐halophilic extremophiles induces the splicing reaction at lower salt concentrations, especially when coupled with a change of the C‐terminal Gln to the more highly conserved Asn. As few as two residue changes can dramatically alter the salt‐dependence of the activity, showing that subtle changes to surface residues can result in conditional folding and activity.

Long-range electrostatic interactions significantly modulate the affinity of dynein for microtubules

The dynein family of microtubule minus-end-directed motor proteins drives diverse functions in eukaryotic cells, including cell division, intracellular transport, and flagellar beating. Motor protein processivity, which characterizes how far a motor walks before detaching from its filament, depends on the interaction between its microtubule-binding domain (MTBD) and the microtubule. Dynein’s MTBD switches between high- and low-binding affinity states as it steps. Significant structural and functional data show that specific salt bridges within the MTBD and between the MTBD and the microtubule govern these affinity state shifts. However, recent computational work suggests that nonspecific, long-range electrostatic interactions between the MTBD and the microtubule may also play an important role in the processivity of dynein. To investigate this hypothesis, we mutated negatively charged amino acids remote from the dynein MTBD-microtubule-binding interface to neutral residues and measured the binding affinity using microscale thermophoresis and optical tweezers. We found a significant increase in the binding affinity of the mutated MTBDs for microtubules. Furthermore, we found that charge screening by free ions in solution differentially affected the binding and unbinding rates of MTBDs to microtubules. Together, these results demonstrate a significant role for long-range electrostatic interactions in regulating dynein-microtubule affinity. Moreover, these results provide insight into the principles that potentially underlie the biophysical differences between molecular motors with various processivities and protein-protein interactions more generally.

Reductive leaching of indium and zinc from zinc neutral leach residue with sulphuric acid and sulphur dioxide

ABSTRACT Indium and zinc extractions from zinc neutral leach residue were investigated with sulphuric acid and sulphur dioxide. Sulphur dioxide maintained a low concentration of Fe3+ in the solution and promoted the decomposition of zinc ferrite. More zinc, indium, and iron dissolve into the solution, while copper co-precipitates with arsenic and sulphur into the residue. The optimal reductive leaching conditions were determined as 120 g/L H2SO4, 200 kPa sulphur dioxide partial pressure, a liquid-to-solid ratio of 8 mL/g and a stirring speed of 500 r/min at 105°C for 4 h. Extractions of 95.24% of zinc, 98.66% of iron, 95.04% of indium and 0.036% of copper were achieved. Copper in final residue exist as sulphides and can be recovered by subsequent oxygen pressure leaching. Selective recovery of valuable metals from zinc neutral leach residue is realised.