Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR.

Modulating allosteric coupling offers unique opportunities for biomedical applications. Such efforts can benefit from efficient prediction and evaluation of allostery hotspot residues that dictate the degree of cooperativity between distant sites. We demonstrate that effects of allostery hotspot mutations can be evaluated qualitatively and semiquantitatively by molecular dynamics simulations in a bacterial tetracycline repressor (TetR). The simulations recapitulate the effects of these mutations on abolishing the induction function of TetR and provide a rationale for the different rescuabilities observed to restore allosteric coupling of the hotspot mutations. We demonstrate that the same noninducible phenotype could be the result of perturbations in distinct structural and energetic properties of TetR. Our work underscores the value of explicitly computing the functional free energy landscapes to effectively evaluate and rank hotspot mutations despite the prevalence of compensatory interactions and therefore provides quantitative guidance to allostery modulation for therapeutic and engineering applications.

Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K+ channel.

Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4-S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.

Second-Shell Residues Contribute to Catalysis by Predominately Preorganizing the Apo State in PafA.

Residues beyond the first coordination shell are often observed to make considerable cumulative contributions in enzymes. Due to typically indirect perturbations of multiple physicochemical properties of the active site, however, their individual and specific roles in enzyme catalysis and disease-causing mutations remain difficult to predict and understand at the molecular level. Here we analyze the contributions of several second-shell residues in phosphate-irrepressible alkaline phosphatase of flavobacterium (PafA), a representative system as one of the most efficient enzymes. By adopting a multifaceted approach that integrates quantum-mechanical/molecular-mechanical free energy computations, molecular-mechanical molecular dynamics simulations, and density functional theory cluster model calculations, we probe the rate-limiting phosphoryl transfer step and structural properties of all relevant enzyme states. In combination with available experimental data, our computational results show that mutations of the studied second-shell residues impact catalytic efficiency mainly by perturbation of the apo state and therefore substrate binding, while they do not affect the ground state or alter the nature of phosphoryl transfer transition state significantly. Several second-shell mutations also modulate the active site hydration level, which in turn influences the energetics of phosphoryl transfer. These mechanistic insights also help inform strategies that may improve the efficiency of enzyme design and engineering by going beyond the current focus on the first coordination shell.

Defluorinative 1,3-Dienylation of Fluoroalkyl N-Triftosylhydrazones with Homoallenols.

A silver-catalyzed regioselective defluorinative 1,3-dienylation of trifluoromethyl phenyl N-triftosylhydrazones using homoallenols as 1,3-dienyl sources provides a variety of α-(di)fluoro-ß-vinyl allyl ketones with excellent functional group tolerance in moderate to good yields. The reaction proceeds through a silver carbene-initiated sequential etherification and Claisen type [3,3]-sigmatropic rearrangement cascade. The synthetic utility of this protocol was demonstrated through the downstream synthetic elaboration toward diverse synthetically useful building blocks.

Electronic Polarization Is Essential for the Stabilization and Dynamics of Buried Ion Pairs in Staphylococcal Nuclease Mutants.

Buried charged residues play important roles in the modulation of protein stabilities and conformational dynamics and make crucial contributions to protein functions. Considering the generally nonpolar nature of protein interior, a key question concerns the contribution of electronic polarization to the stabilization and properties of buried charges. We answer this question by conducting free energy simulations using the latest polarizable CHARMM force field based on Drude oscillators for a series of Staphylococcal nuclease mutants that involve a buried Glu-Lys pair in different titration states and orientations. While a nonpolarizable model suggests that the ionized form of the buried Glu-Lys pair is more than 40 kcal/mol less stable than the charge-neutral form, the two titration states are comparable in stability when electronic polarization is included explicitly, a result better reconcilable with available experimental data. Analysis of free energy components suggests that additional stabilization of the ionized Glu-Lys pair has contributions from both the enhanced salt-bridge strength and stronger interaction between the ion-pair and surrounding protein residues and penetrated water. Despite the stronger direct interaction between Glu and Lys, the ion-pair exhibits considerably larger and faster structural fluctuations when polarization is included, due to compensation of interactions in the cavity. Collectively, observations from this work provide compelling evidence that electronic polarization is essential to the stability, hydration, dynamics, and therefore function of buried charges in proteins. Therefore, our study advocates for the explicit consideration of electronic polarization for mechanistic and engineering studies that implicate buried charged residues, such as enzymes and ion transporters.

Molecular Dynamics Simulations Establish the Molecular Basis for the Broad Allostery Hotspot Distributions in the Tetracycline Repressor.

It is imperative to identify the network of residues essential to the allosteric coupling for the purpose of rationally engineering allostery in proteins. Deep mutational scanning analysis has emerged as a function-centric approach for identifying such allostery hotspots in a comprehensive and unbiased fashion, leading to observations that challenge our understanding of allostery at the molecular level. Specifically, a recent deep mutational scanning study of the tetracycline repressor (TetR) revealed an unexpectedly broad distribution of allostery hotspots throughout the protein structure. Using extensive molecular dynamics simulations (up to 50 µs) and free energy computations, we establish the molecular and energetic basis for the strong anticooperativity between the ligand and DNA binding sites. The computed free energy landscapes in different ligation states illustrate that allostery in TetR is well described by a conformational selection model, in which the apo state samples a broad set of conformations, and specific ones are selectively stabilized by either ligand or DNA binding. By examining a range of structural and dynamic properties of residues at both local and global scales, we observe that various analyses capture different subsets of experimentally identified hotspots, suggesting that these residues modulate allostery in distinct ways. These results motivate the development of a thermodynamic model that qualitatively explains the broad distribution of hotspot residues and their distinct features in molecular dynamics simulations. The multifaceted strategy that we establish here for hotspot evaluations and our insights into their mechanistic contributions are useful for modulating protein allostery in mechanistic and engineering studies.

Efficient Sampling of Cavity Hydration in Proteins with Nonequilibrium Grand Canonical Monte Carlo and Polarizable Force Fields.

Prediction of the hydration levels of protein cavities and active sites is important to both mechanistic analysis and ligand design. Due to the unique microscopic environment of these buried water molecules, a polarizable model is expected to be crucial for an accurate treatment of protein internal hydration in simulations. Here we adapt a nonequilibrium candidate Monte Carlo approach for conducting grand canonical Monte Carlo simulations with the Drude polarizable force field. The GPU implementation enables the efficient sampling of internal cavity hydration levels in biomolecular systems. We also develop an enhanced sampling approach referred to as B-walking, which satisfies detailed balance and readily combines with grand canonical integration to efficiently calculate quantitative binding free energies of water to protein cavities. Applications of these developments are illustrated in a solvent box and the polar ligand binding site in trypsin. Our simulation results show that including electronic polarization leads to a modest but clear improvement in the description of water position and occupancy compared to the crystal structure. The B-walking approach enhances the range of water sampling in different chemical potential windows and thus improves the accuracy of water binding free energy calculations.

Unlocking Maximum Synergy: Screen-Printing Fabrication of Heterostructured Microsupercapacitor Stacks.

A cost-effective and scalable approach for the fabrication of heterostructured microsupercapacitors (MSCs) employing screen-printing followed by sequential electrochemical and microspray deposition techniques has been demonstrated. The microsupercapacitor electrode (MSC) that composed of stacked layers of mesoporous carbon, polyaniline (PANI), and MXene hold significant promise for wearable electronics. By adjusting the deposition and spray cycles, the MSC can be readily coated with PANI and MXene. The sequentially stacked two layers of MXene and PANI on the mesoporous carbon spheres (PMPM-MSC) yielded a specific capacitance of 1003 mF cm-2 at 0.5 mA cm-2, surpassing the performance of PANI/mesoporous carbon electrode by 1.6 times (771 mF cm-2). After 10,000 cycles of charge and discharge, PMPM-MSCs retained more than 86% of their initial capacitance. In-situ Raman spectroscopy confirmed the synergistic effects between MXene and PANI within the heterostructured stacked PMPM-MSC electrodes, including enhanced electronic conductivity and improved electrolyte ion dissociation, which aligned with the electrochemical measurement results, such as fast charge/discharge rates and reduced internal and mass transport resistance. This study demonstrates the potential of screen-printed heterostructured MSC stacks with maximum electrochemical synergy for portable and wearable energy storage devices.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR.

Modulating allosteric coupling offers unique opportunities for biomedical applications. Such efforts can benefit from efficient prediction and evaluation of allostery hotspot residues that dictate the degree of co-operativity between distant sites. We demonstrate that effects of allostery hotspot mutations can be evaluated qualitatively and semi-quantitatively by molecular dynamics simulations in a bacterial tetracycline repressor (TetR). The simulations recapitulate the effects of these mutations on abolishing the induction function of TetR and provide a rationale for the different degrees of rescuability observed to restore allosteric coupling of the hotspot mutations. We demonstrate that the same non-inducible phenotype could be the result of perturbations in distinct structural and energetic properties of TetR. Our work underscore the value of explicitly computing the functional free energy landscapes to effectively evaluate and rank hotspot mutations despite the prevalence of compensatory interactions, and therefore provide quantitative guidance to allostery modulation for therapeutic and engineering applications.

Effects of Dietary Supplementation of Chinese Yam Polysaccharide on Carcass Composition, Meat Quality, and Antioxidant Capacity in Broilers.

The study aimed to evaluate the influences of the dietary supplementation of Chinese yam polysaccharide (CYP) on the carcass performance, antioxidant capacity, and meat quality of broilers. Three hundred and sixty healthy 1-day-old broilers with similar body weight (39 ± 1 g, gender balanced) were randomly divided into four groups (control, CYP1, CYP2, and CYP3 groups). In the control group, broilers were fed a basal diet with CYP, and the CYP1, CYP2, and CYP3 groups were fed diets supplemented with 250, 500, and 1000 mg/kg CYP, respectively. There were three replicates in each group, 30 birds in each replicate, and the feeding trial lasted for 48 days. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) by one-way analysis of variance. The results showed that compared with the control group, dietary supplementation with 500 mg/kg CYP can improve live weight, half-eviscerated carcass percentage, eviscerated carcass percentage, and thigh muscle percentage. Moreover, dietary supplementation with 500 mg/kg CYP can improve the contents of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GPX), and glutathione s-transferase (GST) in serum (p < 0.05). Meanwhile, the mRNA expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), quinone oxidoreductase (NQO1), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), and catalase (CAT) in the liver; the mRNA expression levels of HO-1, NQO1, GPX1, and CAT in the breast muscle; and the mRNA expression levels of NQO1, SOD1, and CAT in the thigh muscle of broilers in the CYP2 group were significantly increased (p < 0.05). In addition, the yellowness and shear force of the thigh and breast muscles and the content of malondialdehyde (MDA) in the serum of broilers in the control group were higher than that in the CYP2 groups (p < 0.05). The results demonstrated that the CYP2 group had the best effect on improving meat quality. In conclusion, dietary supplementation with 500 mg/kg CYP can improve the meat quality of broilers by improving carcass quality, meat color, shear force, and antioxidant capacity.

Effects of dietary Chinese yam polysaccharide copper complex on growth performance, immunity, and antioxidant capacity of broilers.

Chinese yam polysaccharide (CYP) has received attention in recent years owing to its positive nutritional and medicinal characteristics. Copper is an essential trace metal in animals, which plays an important role in iron absorption and hemoglobin synthesis. However, no published study has evaluated Chinese yam polysaccharide copper complex (CYP-Cu) as a dietary additive in broilers. This study was conducted to investigate the effects of dietary CYP-Cu on growth performance, immunity, and oxidative resistance in broilers. A total of 360 1-day-old 817 broiler chickens were randomly divided into 4 groups, with 3 replicates of 30 birds each and were fed a basal diet with the addition of 0 (control group), 0.02, 0.10, and 0.50 g/kg CYP-Cu. The feeding trial lasted 48 days. On day 28 and day 48, 6 broilers in each group were slaughtered, respectively. Then the parameters of growth and carcass, serum biochemistry, immunity, and antioxidation, and the expression level of hepatic antioxidative genes were investigated. The results showed that compared with the control group, the supplementation of dietary CYP-Cu could improve the indexes of the growth, carcass, serum biochemistry, immunity and oxidation resistance in broilers, such as average daily gain (ADG), the slaughter percentage (SP), semi-evisceration weight percentage (SEWP), eviscerated carcass weight percentage (EWP), breast muscle percentage (BMP), leg muscle percentage (LMP), serum albumin (ALB), high density lipoprotein (HDL), insulin-like growth factor I (IGF-I), triiodothyronine (T3), thyroxine (T4), growth hormone (GH), insulin (INS), immunoglobulin M (IgM), immunoglobulin G (IgG), immunoglobulin A (IgA), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), complement 3 (C3), complement 4 (C4), total superoxide dismutase (T-SOD), total antioxidative capacity (T-AOC), glutathione peroxidase (GSH-Px), and glutathione s-transferase (GSH-ST); these parameters in the 0.10 g/kg CYP-Cu treated group were significantly increased (P < 0.05) in the total trial period, with the exceptions that feed conversion ratio (FCR) and serum low density lipoprotein (LDL), malondialdehyde (MDA) were decreased in the total trial period. In addition, the antioxidative gene mRNA expression of Nuclear factor E2-related factor 2 (Nrf 2), Superoxide dismutase 1 (SOD 1), Superoxide dismutase 2 (SOD 2), and Catalase (CAT) were upregulated in the liver (P < 0.05). These results indicated that the supplementation of dietary CYP-Cu improved the growth, immunity, and oxidation resistance of broilers, and the addition of 0.10 g/kg CYP-Cu in broiler diets is recommended, which suggests that CYP-Cu may be a promising green feed additive in the poultry industry.

Effects of Chinese Yam Polysaccharide on Intramuscular Fat and Fatty Acid Composition in Breast and Thigh Muscles of Broilers.

The purpose of this study is to evaluate the influences of Chinese yam polysaccharide (CYP) dietary supplementation on the composition of intramuscular fat (IMF) and fatty acids (FA) in thigh and breast muscles of broilers. Three hundred and sixty healthy one-day-old broilers (the breed of Crossbred chicken is named 817) with gender-balanced and similar body weight (39 ± 1 g) were randomly allocated into four groups (control, CYP1, CYP2, and CYP3 groups). Broilers in the control group were only fed a basal diet, and broilers in CYP1 group were fed the same diets further supplemented with 250 mg/kg CYP, the CYP2 group was fed the same diets further supplemented with 500 mg/kg CYP, and the CYP3 group was fed the same diets further supplemented with 1000 mg/kg CYP, respectively. Each group consisted of three replicates and each replicate consisted of 30 birds. The feeding days were 48 days. The results observed that the CYP2 group (500 mg/kg) can up-regulate the mRNA expression levels of ß-catenin in thigh muscle compared to the control group. At the same time, all CYP groups (CYP1, CYP2, and CYP3 groups) can up-regulate mRNA expression of Wnt1 and ß-catenin in breast muscle, while mRNA expression of PPARγ and C/EBPα in breast and thigh muscles could be down-regulated (p < 0.05). In summary, 500 mg/kg of CYP dietary supplementation can reduce IMF content and improve the FAs composition, enhancing the nutritional value of chicken meat.

Rescue intracranial stenting for acute ischemic stroke after the failure of mechanical thrombectomy: A systematic review, meta-analysis, and trial sequential analysis.

Background: Intracranial rescue stenting (RS) might be an option for acute ischemic stroke after the failure of mechanical thrombectomy (MT). However, the findings were not consistent in previous systematic reviews, and whether the conclusion was supported by sufficient statistical power is unknown. Aim: To examine the effect of RS on acute ischemic stroke after the failure of MT with a systematic review, meta-analysis, and trial sequential analysis (TSA). Methods: We searched Ovid Medline, Embase, and the Cochrane Central Register of Controlled Trials (CENTRAL) from inception to 15 June 2022, without any language restriction. Studies assessing the effect of RS for acute ischemia stroke after MT failure were included. Two reviewers independently screened the retrieved articles, extracted data, and evaluated the quality of the included studies through the New Ottawa Scale (NOS). The primary outcome was the recanalization rate after RS. Secondary outcomes included modified Rankin Scale (mRS) at 3 months after stroke, symptomatic intracranial hemorrhage (sICH), and mortality rate. We synthesized the data through a random-effects model and performed a TSA analysis. Results: We included 15 studies (containing 1,595 participants) after screening 3,934 records. The pooled recanalization rate for rescue stenting was 82% (95% CI 77-87%). Compared with non-stenting, rescue stenting was associated with a higher proportion of patients with 0-2 mRS score (OR 3.96, 95% CI 2.69-5.84, p < 0.001) and a lower 90-day mortality rate (OR 0.46, 95% CI 0.32-0.65, p < 0.001), and stenting did not increase sICH rate (OR 0.63, 95% CI 0.39-1.04, p = 0.075). The TSA analysis showed that the meta-analysis of the mRS score had a sufficient sample size and statistical power. Conclusions: Our study showed that rescue stenting was effective and safe for patients with acute ischemia stroke who also had a failed MT, and this result was confirmed in a TSA analysis.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on mRNA in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a bisubstrate analogue representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release catalysed by METTL3, and suggests that the latter step is rate-limiting. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Effects of Chinese yam polysaccharides on the immune function and serum biochemical indexes of broilers.

The purpose of this experiment was to investigate the effects of Chinese yam polysaccharides (CYP) in diets on the immune function of broilers. A total of 360 (1-day-old, sex balance) healthy growing broilers with similar body weight (39.54 ± 0.51 g) were randomly divided into control (0.00 g/kg), CYP I (0.25 g/kg), CYP II (0.50 g/kg), and CYP III (1.00 g/kg) groups. Each group contains 3 replicates with 30 broilers in each replicate, and the feeding trial lasted 48 d. The results showed that compared with the control group, the CYP II group had higher thymus index, serum IgA, complement C3, C4, IGF-I, T3, T4, INS, GH, IL-2, IL-4, IL-6, and TNF-α levels (P < 0.05) at 28, 48 d, respectively. In addition, the spleen index, serum IgM and IgG concentrations in CYP II group were higher than those in the control group at 28 d (P < 0.05). Results indicated that 0.50 g/kg CYP supplementation improved the immune function of broilers, and the CYP has a potential biological function as a green additive in broilers.

Effects of Chinese yam Polysaccharides on the Muscle Tissues Development-Related Genes Expression in Breast and Thigh Muscle of Broilers.

This experiment was conducted to evaluate the effects of dietary Chinese yam polysaccharides (CYP) on myogenic differentiation 1 (MYOD1), myogenin (MYOG), and myostatin (MSTN) mRNA expression of breast and thigh muscle tissues in broilers. A total of 360 (1-day-old, gender-balanced) crossbred broilers chicks with similar body weight (BW) were randomly distributed into four groups, with three replicates in each group and each replicate included 30 broilers. The feeding trial lasted for 48 days. Experimental broilers were fed 0.00 mg/kg basal diet (control group), 250 mg/kg, 500 mg/kg, and 1000 mg/kg CYP, respectively. The results showed that CYP250 and CYP500 groups had higher thigh muscle percentage (TMP) compared to the control group (p < 0.05). Meanwhile, the expression of MYOD1, MYOG mRNA in breast muscle tissues of CYP500 and CYP1000 groups was higher (p < 0.05), and the expression of MSTN mRNA in thigh muscle of CYP250, CYP500, and CYP1000 groups was lower than that of the control group (p < 0.05). In addition, there was no significant difference in the expression of MYOD1 mRNA in the thigh muscle tissue of each group (p > 0.05). Bivariate correlation analysis showed that the expression levels of MYOD1, MYOG, and MSTN mRNA in the thigh muscle tissue of broiler chickens in the CYP500 group were positively correlated with TMP. However, the expression of MYOG mRNA in thigh muscle tissue of the CYP1000 group was negatively correlated with TMP. In general, this study indicated that appropriate dietary CYP supplementation influenced the growth and development of thigh muscle tissue in broilers by altering TMP and muscle tissue development-related genes expression. Therefore, CYP could be used as a potential feed additive to promote the development of muscle tissues in broilers.

Reverse Protonation of Buried Ion-Pairs in Staphylococcal Nuclease Mutants.

Although buried titratable residues in protein cavities are often of major functional importance, it is generally challenging to understand their properties such as the ionization state and factors of stabilization based on experimental studies alone. A specific set of examples involve buried Glu-Lys pairs in a series of variants of Staphylococcal nuclease, for which recent structural and thermodynamic studies appeared to suggest that both the stability and the ionization state of the buried Glu-Lys pair are sensitive to its orientation (i.e., Glu23-Lys36 vs Lys23-Glu36). To further clarify the situation, especially ionization states of the buried Glu-Lys pairs, we have conducted extensive molecular dynamics simulations and free energy computations. Microsecond molecular dynamics simulations show that the hydration level of the cavity depends on the orientation of the buried ion-pair therein as well as its ionization state; free energy simulations recapitulate the relative stability of Glu23-Lys36 (EK) vs Lys23-Glu36 (KE) mutants measured experimentally, although the difference is similar in magnitude regardless of the ionization state of the Glu-Lys pair. A complementary set of free energy simulations strongly suggests that, in contrast to the original suggestion in the experimental analysis, the Glu and Lys residues prefer to adopt their charge-neutral rather than the ionized states. This result is consistent with the low dielectric constant computed for water in the cavity, which makes it difficult for the protein cavity to stabilize a pair of charged Glu-Lys residues, even with water penetration. The current study highlights the role of free energy simulations in understanding the ionization state of buried titratable residues and the relevant energetic contributions, forming the basis for the rational design of buried charge networks in proteins.

Genomic analysis of porcine circovirus type 2 from southern China.

BACKGROUND: Porcine circovirus type 2 (PCV2) is recognized as virulent porcine pathogen and has been linked to porcine circovirus diseases (PCVD). However, there remain many unknowns regarding the spread and epidemic growth of PCV2. METHODS: To assess the genetic diversity of PCV2 in the southern China, a total of 92 sequences of PCV2 strains from this region were retrieved from GenBank and were subjected to amino acid variation and phylogenetic analyses together with 28 representative sequences, based on the sequence of the ORF2 gene, from different swine-producing countries. RESULTS: All 92 PCV2 strains shared between 93.7% and 100% sequence similarity and could be divided into four genotypes (PCV2a, PCV2b, PCV2d and PCV2h), of which PCV2d had surpassed PCV2b and became the most prevalent PCV2 genotype in this region. Alignment of the deduced amino acid sequences of the capsid protein revealed that the obtained PCV2 strains possess two major heterogenic regions/hypervariable regions (positions 52-68 and 185-191), which were within or close to the epitopic regions in the capsid (Cap) protein. Meanwhile, the 92 PCV2 sequences also show evidence of at least five unique recombination events. CONCLUSION: The data in this study indicate that the PCV2 strains in the southern China are undergoing constant genetic variation and that the predominant strain and its antigenic epitopes in this area have been gradually changing in recent years.

Multicolor polymeric carbon dots: synthesis, separation and polyamide-supported molecular fluorescence.

Multicolor carbon dots (CDs) have been developed recently and demonstrate great potential in bio-imaging, sensing, and LEDs. However, the fluorescence mechanism of their tunable colors is still under debate, and efficient separation methods are still challenging. Herein, we synthesized multicolor polymeric CDs through solvothermal treatment of citric acid and urea in formamide. Automated reversed-phase column separation was used to achieve fractions with distinct colors, including blue, cyan, green, yellow, orange and red. This work explores the physicochemical properties and fluorescence origins of the red, green, and blue fractions in depth with combined experimental and computational methods. Three dominant fluorescence mechanism hypotheses were evaluated by comparing time-dependent density functional theory and molecular dynamics calculation results to measured characteristics. We find that blue fluorescence likely comes from embedded small molecules trapped in carbonaceous cages, while pyrene analogs are the most likely origin for emission at other wavelengths, especially in the red. Also important, upon interaction with live cells, different CD color fractions are trafficked to different sub-cellular locations. Super-resolution imaging shows that the blue CDs were found in a variety of organelles, such as mitochondria and lysosomes, while the red CDs were primarily localized in lysosomes. These findings significantly advance our understanding of the photoluminescence mechanism of multicolor CDs and help to guide future design and applications of these promising nanomaterials.