Dynamic Allostery in the Methionine Repressor Revealed by Force Distribution Analysis

Many fundamental cellular processes such as gene expression are tightly regulated by protein allostery. Allosteric signal propagation from the regulatory to the active site requires long-range communication, the molecular mechanism of which remains a matter of debate. A classical example for long-range allostery is the activation of the methionine repressor MetJ, a transcription factor. Binding of its co-repressor SAM increases its affinity for DNA several-fold, but has no visible conformational effect on its DNA binding interface. Our molecular dynamics simulations indicate correlated domain motions within MetJ, and quenching of these dynamics upon SAM binding entropically favors DNA binding. From monitoring conformational fluctuations alone, it is not obvious how the presence of SAM is communicated through the largely rigid core of MetJ and how SAM thereby is able to regulate MetJ dynamics. We here directly monitored the propagation of internal forces through the MetJ structure, instead of relying on conformational changes as conventionally done. Our force distribution analysis successfully revealed the molecular network for strain propagation, which connects collective domain motions through the protein core. Parts of the network are directly affected by SAM binding, giving rise to the observed quenching of fluctuations. Our results are in good agreement with experimental data. The force distribution analysis suggests itself as a valuable tool to gain insight into the molecular function of a whole class of allosteric proteins

Reorganization Energy for Internal Electron Transfer in Multicopper Oxidases.

We have calculated the reorganization energy for the intramolecular electron transfer between the reduced type 1 copper site and the peroxy intermediate of the trinuclear cluster in the multicopper oxidase CueO. The calculations are performed at the combined quantum mechanics and molecular mechanics (QM/MM) level, based on molecular dynamics simulations with tailored potentials for the two copper sites. We obtain a reorganization energy of 91-133 kJ/mol, depending on the theoretical treatment. The two Cu sites contribute by 12 and 22 kJ/mol to this energy, whereas the solvent contribution is 34 kJ/mol. The rest comes from the protein, involving small contributions from many residues. We have also estimated the energy difference between the two electron-transfer states and show that the reduction of the peroxy intermediate is exergonic by 43-87 kJ/mol, depending on the theoretical method. Both the solvent and the protein contribute to this energy difference, especially charged residues close to the two Cu sites. We compare these estimates with energies obtained from QM/MM optimizations and QM calculations in a vacuum and discuss differences between the results obtained at various levels of theory

Catalytic Cycle of Multicopper Oxidases Studied by Combined Quantum- and Molecular-Mechanical Free-Energy Perturbation Methods

We have used combined quantum mechanical and molecular mechanical free-energy perturbation methods in combination with explicit solvent simulations to study the reaction mechanism of the multicopper oxidases, in particular the regeneration of the reduced state from the native intermediate. For 52 putative states of the trinuclear copper cluster, differing in the oxidation states of the copper ions and the protonation states of water- and O2-derived ligands, we have studied redox potentials, acidity constants, isomerisation reactions, as well as water- and O2 binding reactions. Thereby, we can propose a full reaction mechanism of the multicopper oxidases with atomic detail. We also show that the two copper sites in the protein communicate so that redox potentials and acidity constants of one site are affected by up to 0.2 V or 3 pKa units by a change in the oxidation state of the other site

Environment influences on the aromatic character of nucleobases and amino acids

Geometric (HOMA) and magnetic (NICS) indices of aromaticity were estimated for aromatic rings of amino acids and nucleobases. Cartesian coordinates were taken directly either from PDB files deposited in public databases at the finest resolution available (≤1.5 Å), or from structures resulting from full gradient geometry optimization in a hybrid QM/MM approach. Significant environmental effects imposing alterations of HOMA values were noted for all aromatic rings analysed. Furthermore, even extra fine resolution (≤1.0 Å) is not sufficient for direct estimation of HOMA values based on Cartesian coordinates provided by PDB files. The values of mean bond errors seem to be much higher than the 0.05 Å often reported for PDB files. The use of quantum chemistry geometry optimization is strongly advised; even a simple QM/MM model comprising only the aromatic substructure within the QM region and the rest of biomolecule treated classically within the MM framework proved to be a promising means of describing aromaticity inside native environments. According to the results presented, three consequences of the interaction with the environment can be observed that induce changes in structural and magnetic indices of aromaticity. First, broad ranges of HOMA or NICS values are usually obtained for different conformations of nearest neighborhood. Next, these values and their means can differ significantly from those characterising isolated monomers. The most significant increase in aromaticities is expected for the six-membered rings of guanine, thymine and cytosine. The same trend was also noticed for all amino acids inside proteins but this effect was much smaller, reaching the highest value for the five-membered ring of tryptophan. Explicit water solutions impose similar changes on HOMA and NICS distributions. Thus, environment effects of protein, DNA and even explicit water molecules are non-negligible sources of aromaticity changes appearing in the rings of nucleobases and aromatic amino acids residues

Characterization of the thermal and photoinduced reactions of photochromic spiropyrans in aqueous solution

Six water-soluble spiropyran derivatives have been characterized with respect to the thermal and photoinduced reactions over a broad pH-interval. A comprehensive kinetic model was formulated including the spiro- and the merocyanine isomers, the respective protonated forms, and the hydrolysis products. The experimental studies on the hydrolysis reaction mechanism were supplemented by calculations using quantum mechanical (QM) models employing density functional theory. The results show that (1) the substitution pattern dramatically influences the pKa-values of the protonated forms as well as the rates of the thermal isomerization reactions, (2) water is the nucleophile in the hydrolysis reaction around neutral pH, (3) the phenolate oxygen of the merocyanine form plays a key role in the hydrolysis reaction. Hence, the nonprotonated merocyanine isomer is susceptible to hydrolysis, whereas the corresponding protonated form is stable toward hydrolytic degradation

An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl− system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall

Evidence and gap map of studies assessing the effectiveness of interventions for people with disabilities in low‐and middle‐income countries

Background: There are approximately 1 billion people in the world with some form of disability. This corresponds to approximately 15% of the world's population (World Report on Disability, 2011). The majority of people with disabilities (80%) live in low- and middle-income countries (LMICs), where disability has been shown to disproportionately affect the most disadvantaged sector of the population. Decision makers need to know what works, and what does not, to best invest limited resources aimed at improving the well-being of people with disabilities in LMICs. Systematic reviews and impact evaluations help answer this question. Improving the availability of existing evidence will help stakeholders to draw on current knowledge and to understand where new research investments can guide decision-making on appropriate use of resources. Evidence and gap maps (EGMs) contribute by showing what evidence there is, and supporting the prioritization of global evidence synthesis needs and primary data collection. Objectives: The aim of this EGM is to identify, map and describe existing evidence of effectiveness studies and highlight gaps in evidence base for people with disabilities in LMICs. The map helps identify priority evidence gaps for systematic reviews and impact evaluations. Methods: The EGM included impact evaluation and systematic reviews assessing the effect of interventions for people with disabilities and their families/carers. These interventions were categorized across the five components of community-based rehabilitation matrix; health, education, livelihood, social and empowerment. Included studies looked at outcomes such as, health, education, livelihoods, social inclusion and empowerment, and were published for LMICs from 2000 onwards until January 2018. The searches were conducted between February and March 2018. The EGM is presented as a matrix in which the rows are intervention categories (e.g., health) and subcategories (e.g., rehabilitation) and the column outcome domains (e.g., health) and subdomains (e.g., immunization). Each cell lists the studies for that intervention for those outcomes, with links to the available studies. Included studies were therefore mapped according to intervention and outcomes assessed and additional filters as region, population and study design were also coded. Critical appraisal of included systematic review was done using A Measurement Tool to Assess Systematic Reviews’ rating scale. We also quality-rated the impact evaluation using a quality assessment tool based on various approaches to risk of bias assessment. Results: The map includes 166 studies, of which 59 are systematic reviews and 107 impact evaluation. The included impact evaluation are predominantly quasiexperimental studies (47%). The numbers of studies published each year have increased steadily from the year 2000, with the largest number published in 2017.The studies are unevenly distributed across intervention areas. Health is the most heavily populated area of the map. A total of 118 studies of the 166 studies concern health interventions. Education is next most heavily populated with 40 studies in the education intervention/outcome sector. There are relatively few studies for livelihoods and social, and virtually none for empowerment. The most frequent outcome measures are health-related, including mental health and cognitive development (n = 93), rehabilitation (n = 32), mortality and morbidity (n = 23) and health check-up (n = 15). Very few studies measured access to assistive devices, nutrition and immunization. Over half (n = 49) the impact evaluation come from upper-middle income countries. There are also geographic gaps, most notably for low income countries (n = 9) and lower-middle income countries (n = 34). There is a fair amount of evidence from South Asia (n = 73) and Sub-Saharan Africa (n = 51). There is a significant gap with respect to study quality, especially with respect to impact evaluation. There appears to be a gap between the framing of the research, which is mostly within the medical model and not using the social model of disability. Conclusion: Investing in interventions to improve well-being of people with disabilities will be critical to achieving the 2030 agenda for sustainable development goals. The EGM summarized here provides a starting point for researchers, decision makers and programme managers to access the available research evidence on the effectiveness of interventions for people with disabilities in LMICs in order to guide policy and programme activity, and encourage a more strategic, policy-oriented approach to setting the future research agenda