Redox Potentials of Protein Disulfide Bonds from Free-Energy Calculations

Thiol/disulfide exchange in proteins is a vital process in all organisms. To ensure specificity, the involved thermodynamics and kinetics are believed to be tailored by the structure and dynamics of the protein hosting the thiol/disulfide pair. We here aim at predicting the thermodynamics of thiol/disulfide pairs in proteins. We devise a free-energy calculation scheme, which makes use of the Crooks Gaussian intersection method to estimate the redox potential of thiol/disulfide pairs in 12 proteins belonging to the thioredoxin superfamily, namely, thioredoxins, glutaredoxins, and thiol–disulfide oxidoreductases in disulfide bond formation systems. We obtained a satisfying correlation of computed with experimental redox potentials (varying by 160 mV), with a residual error of ∼40 mV (8 kJ/mol), which drastically reduces when considering a less diverse set of only thioredoxins. Our simple and transferrable approach provides a route toward estimating redox potentials of any disulfide-containing protein given that its (reduced or oxidized) structure is known and thereby represents a step toward a rational design of redox proteins

Exploring the Multidimensional Free Energy Surface of Phosphoester Hydrolysis with Constrained QM/MM Dynamics

The mechanism of the hydrolysis of phosphate monoesters, a ubiquitous biological reaction, has remained under debate. We here investigated the hydrolysis of a nonenzymatic model system, the monomethyl phosphate dianion, by hybrid quantum mechanical and molecular mechanical simulations. The solvation effects were taken into account with explicit water. Detailed free energy landscapes in two-dimensional and three-dimensional space were resolved using the multidimensional potential of mean constraint force, a newly developed method that was demonstrated to be powerful for free energy calculations along multiple coordinates. As in previous theoretical studies, the associative and dissociative pathways were indistinguishable. Furthermore, the associative pathway was investigated in great detail. We propose a rotation of an O–H bond in the transition between two pentacoordinated structures, during which an overall transition state was identified with an activation energy of 50 kcal/mol. This is consistent with experimental data. The results support a concerted proton transfer from the nucleophilic water to the phosphate group, and then to the leaving group

Functional group dominance and not productivity drives species richness

<p><b><i>Background</i></b>: There is a lack of consensus about the productivity–richness relationship, with several recent studies suggesting that it is not productivity but other factors that are the important drivers that determine species richness.</p> <p><b><i>Aims</i></b>: Here, we examine the relationship between productivity, functional group dominance and plant species richness at the plot scale in Tibetan Plateau meadows. These alpine meadows are ideal to examine the species productivity-richness relationship because they have a very high species richness, a large gradient in productivity, and can be dominated by either graminoids (grasses and sedges) or forbs.</p> <p><b><i>Methods</i></b>: We measured plant species richness and above-ground biomass along a natural gradient of functional group abundance in 44 plots distributed across five natural, winter-grazed but otherwise undisturbed sites in the eastern part of the Qing-Hai Tibetan Plateau, in Gansu province, China in 2008.</p> <p><b><i>Results</i></b>: Graminoid abundance (i.e. graminoid biomass as percent of the total above-ground biomass) explained 39% of plot differences in species richness while neither productivity nor the biomass of the three most abundant plant species, either individually or combined, were a significant predictor of species richness.</p> <p><b><i>Conclusions</i></b>: Our results show that within these alpine meadows, a shift from graminoid to forb dominance, rather than the individual dominant species or productivity itself, is strongly correlated with species richness. Thus, differences in functional group abundance can be a strong driver of observed plant species richness patterns.</p

Association between the variability of the <i>ABCA13</i> gene and the risk of major depressive disorder and schizophrenia in the Han Chinese population

<p><b>Objectives:</b> The ATP-binding cassette transporter superfamily is one of the largest membrane protein families, which is responsible for transportation of substances across the membranes by utilising energy. Some research has bridged the variations in <i>ABCA13</i> with occurrence of psychiatric disorders. To investigate the overlapping risk conferred by <i>ABCA13</i> for both major depressive disorder and schizophrenia, we analysed tag single nucleotide polymorphisms (tag SNPs).</p> <p><b>Methods:</b> We used TaqMan^® technology to genotype 1045 major depressive disorder patients, 1235 schizophrenia patients and 1235 healthy controls of Han Chinese origin.</p> <p><b>Results:</b> We found that rs7789493 (<i>P</i>_allele=7.23E-04, <i>P</i>_genotype=.001) was associated with major depressive disorder, while rs17132388 (<i>P</i>_allele=1.63E-04, <i>P</i>_genotype=7.50E-04) and rs6583476 (<i>P</i>_allele=5.50E-04, <i>P</i>_genotype=.002) showed statistically significant association with schizophrenia.</p> <p><b>Conclusions:</b> Our results indicate that the <i>ABCA13</i> gene may contain overlapping common genetic risk factors for both major depressive disorder and schizophrenia in the Han Chinese population. The study on variants conferring overlapping risk for multiple psychiatric disorders could be tangible pathogenesis support in clinical or diagnostic references.</p

Mapping of MnaA LOF mutations into the MnaA crystal structure reveal key residues for substrate binding site stability and charge.

<p>(A) Overall MRSA COL MnaA crystal structure. The molecular surface is shown in grey. The protein is represented as a cartoon. In all figures one monomer is consistently colored in orange and the other in cyan, and the bound UDP molecules are shown as sticks, methyl groups colored in light blue. Nitrogen, oxygen and phosphor atoms are in blue, red or orange, respectively. (B,C,D,E) Comparison with the <i>M</i>. <i>jannaschii</i> structure in “opened” form (PDB 3NEQ) or “closed” form (PDB 3NES). Both structures are represented as ribbons, one monomer at a time, and UDP as sticks. (B) and (C) compares the opened form, in grey, with each monomer, while the superposition is with the closed form, in (D) and (E), drawn in purple. The RMS deviation in Cα positions are 1.6Å for 262 atoms, 1.6Å for 256 atoms, 1.5Å for 321 atoms, and 1.3Å for 336 atoms, for the superpositions in cartoon (B), (C), (D) and (E), respectively. (F) Mapping MRSE LOF mutants. Eight mutation sites are mapped onto the X-ray crystal structure of UDP bound MRSA COL MnaA. The allosteric site ligand UDP-GlcNAc was taken from the structure of UDP-GlcNAc bound <i>B</i>. <i>anthracis</i> 2-epimerase (PDB ID 3BEO). UDP and UDP-GlcNAc are displayed as thin lines with the carbon atoms colored in light blue. One monomer of MnaA dimer is colored in cyan and the other in white. The mutation sites are highlighted in stick. The carbon atoms of the wild-type residues are colored in yellow; those of the mutant residues are in green. (G) Mapping MRSA <i>mnaA</i> LOF mutants. LOF mutations isolated in MRSA COL MnaA are highlighted. All coloring as in (C), but for simplicity, only the original sequence is shown.</p

MRSA and MRSE MnaA LOF mutants are highly susceptible to imipenem in a murine thigh infection.

<p>Immune-suppressed CD-1 mice (5 per group) were challenged intramuscularly with the parental MRSA COL strain, MRSA <i>Δcap5P</i>, or MRSA <i>Δcap5P mnaA</i>_<i>Sa</i> LOF mutants (A) or with the parental MRSE strain versus <i>mnaA</i>_<i>Se</i>, <i>tarO</i>_<i>Se</i> and <i>tarA</i>_<i>Se</i> LOF mutants (B) and treated three times daily (TID) with imipenem (IPM). Thighs were harvested at 24hrs, homogenized and plated to determine CFU per thigh. (A) Restored efficacy of IPM (10 mg kg^-1) against MRSA <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>. Following IPM treatment, bacterial burden amongst mice infected with <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i> is reduced approximately 2–3 log at 24 hrs versus those infected with MRSA COL or <i>Δcap5P</i> controls. * p<0.01 versus parent at 24 hr; $ p<0.05 versus respective 24 hr vehicle. (B) Restored efficacy of IPM (2.5 mg kg^-1) against MRSE <i>mnaA</i>, <i>tarO</i>, and <i>tarA</i> LOF mutants. Reduction in bacterial burden of mice infected with the <i>mnaA</i>_<i>Se</i>^<i>G171D</i> is comparable to those infected with <i>tarO</i>_<i>Se</i>^<i>G84</i>* or <i>tarA</i>_<i>Se</i>^<i>G129R</i> mutants, yielding an approximate 3 log reduction in 24 hr IPM treatment versus the wild-type control. Note, as MRSE CLB26329 is more susceptible to IPM than MRSA COL, its dose was reduced to 4-fold versus the MRSA efficacy study (A).</p

MnaA loss of function mutants in MRSA and MRSE fail to produce WTA.

<p>WTA extraction and SDS PAGE analysis from L638^R MRSE CLB26329 (A) and MRSA COL (B) mutants. Note, wild-type MRSA WTA polymers appear as a ladder of discretely sized bands whereas a more diffuse staining of MRSE WTA polymer is observed. WTA material was normalized to cell biomass prior to loading. Wild-type copies of <i>cap5P</i>, <i>mnaA</i>_<i>Sa</i>, and <i>mnaA</i>_<i>Se</i>, as well as the empty vector introduced into these strains for complementation studies are indicated. The <i>tarO</i> and <i>tarA</i> deletion mutants serve as a control for complete impairment of WTA polymer production.</p

Tunicamycin inhibits MnaA in a concentration-dependent manner.

<p>Representative electropherograms of the MnaA-catalyzed conversion of UDP-ManNAc to UDP-GlcNAc in the absence of tunicamycin (A), in the presence of 100 μM (B) and 200 μM tunicamycin (C). Substrate concentration is 100 μM. Peaks: <b>1</b> UDP-ManNAc; <b>2</b> UDP-GlcNAc.</p

Biophysical studies demonstrate MnaA and Cap5P bind tunicamycin.

<p>(A, D) 600 MHz 1H NMR spectra of 15 μM tunicamycin. (B, E) 1H NMR STD spectra of 15 μM tunicamycin without 2-epimerase. (C) 1H NMR STD spectra of 15 μM tunicamycin in presence of 5 μM MnaA. (F) 1H NMR STD spectra of 15 μM tunicamycin in presence of 5 μM Cap5P. Saturation of the protein was achieved with a Gaussian pulse cascade resulting in a total saturation time of 3s. The protein resonances were saturated at 100 Hz and the off resonance was set to -120 ppm. Tunicamycin-specific peaks in NMR STD spectra were only obtained in the presence of MnaA or Cap5P. (G) Structure of tunicamycin.</p

Whole-genome sequencing of L638^R mutants.

<p>Heat map summary of all non-synonymous mutations identified by Illumina-based whole-genome sequencing (100X genome coverage) of L638^R mutants in MRSE CLB26329 (A) or MRSA COL (B). Red, non-synonymous mutation; grey, no change versus parental genome sequence; yellow, non-synonymous mutations in genes other than <i>mnaA</i>. Genome position, base pair change, and resulting amino acid residue substitution are highlighted. Note: with only one exception (<i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>), no additional non-synonymous mutations besides the indicated <i>mnaA</i> mutation were identified in each of the drug resistant strains examined.</p