Structural Insights Into High Density Lipoprotein: Old Models and New Facts

The physiological link between circulating high density lipoprotein (HDL) levels and cardiovascular disease is well-documented, albeit its intricacies are not well-understood. An improved appreciation of HDL function and overall role in vascular health and disease requires at its foundation a better understanding of the lipoprotein\u27s molecular structure, its formation, and its process of maturation through interactions with various plasma enzymes and cell receptors that intervene along the pathway of reverse cholesterol transport. This review focuses on summarizing recent developments in the field of lipid free apoA-I and HDL structure, with emphasis on new insights revealed by newly published nascent and spherical HDL models constructed by combining low resolution structures obtained from small angle neutron scattering (SANS) with contrast variation and geometrical constraints derived from hydrogen–deuterium exchange (HDX), crosslinking mass spectrometry, electron microscopy, Förster resonance energy transfer, and electron spin resonance. Recently published low resolution structures of nascent and spherical HDL obtained from SANS with contrast variation and isotopic labeling of apolipoprotein A-I (apoA-I) will be critically reviewed and discussed in terms of how they accommodate existing biophysical structural data from alternative approaches. The new low resolution structures revealed and also provided some answers to long standing questions concerning lipid organization and particle maturation of lipoproteins. The review will discuss the merits of newly proposed SANS based all atom models for nascent and spherical HDL, and compare them with accepted models. Finally, naturally occurring and bioengineered mutations in apoA-I, and their impact on HDL phenotype, are reviewed and discuss together with new therapeutics employed for restoring HDL function

The Solvent Boundary Potential: A New Approach for Computer Simulation of Large Systems

The paper presents the theoretical framework for the development of a new solvent boundary method for computer simulations and its possible application to the simulation of nitric oxide synthase. Different approaches for the construction of a solvent boundary potential are reviewed and their strengths and weaknesses are discussed. A new solvent boundary potential is proposed which combines the mean field force approximation with the Green\u27s function approach for treating long-range electrostatic interactions, and introduces a novel strategy to treat electrostriction effects due to ions crossing the solvent boundary. Finally, a series of computational tests are devised in order to assess the validity and efficiency of the new solvent boundary potential

[Fe-Fe]-Hydrogenase Reactivated by Residue Mutations as Bridging Carbonyl Rearranges: A QM/MM Study

In this work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O2, or OH−, which metabolizes to H2O (Dogaru et al., Int J Quantum Chem 2008, 108; Motiu et al., Int J Quantum Chem 2007, 107, 1248). We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results (Liu et al., J Am Chem Soc 2002, 124, 5175), that is, wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fed) has been screened for residues that prevent H2O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs\u27 energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs\u27 energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs\u27 energy for the elimination of H2O. Favorable thermodynamic results have been obtained for both single residue removal (ΔGΔGlu374 = −1.6 kcal/mol), single substitution (ΔGGlu374His = −3.1 kcal/mol), and combined residue substitutions (ΔGArg111Glu;Thr145Val;Glu374His;Tyr375Phe = −7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, that is, for H2O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are ΔArg111, ΔThr145, ΔSer177, ΔGlu240, ΔGlu374, and ΔTyr375. The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed to compensate for the electron density donated to oxygen upon the elimination of H2O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of COb towards Fed, which leads to enzyme inhibition. Nevertheless, enzyme reactivation can be achieved by means of appropriate amino acid substitutions

[Fe-Fe]-Hydrogenase Reactivated by Residue Mutations as Bridging Carbonyl Rearranges: A QM/MM Study

In this work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O2, or OH−, which metabolizes to H2O (Dogaru et al., Int J Quantum Chem 2008, 108; Motiu et al., Int J Quantum Chem 2007, 107, 1248). We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results (Liu et al., J Am Chem Soc 2002, 124, 5175), that is, wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fed) has been screened for residues that prevent H2O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs\u27 energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs\u27 energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs\u27 energy for the elimination of H2O. Favorable thermodynamic results have been obtained for both single residue removal (ΔGΔGlu374 = −1.6 kcal/mol), single substitution (ΔGGlu374His = −3.1 kcal/mol), and combined residue substitutions (ΔGArg111Glu;Thr145Val;Glu374His;Tyr375Phe = −7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, that is, for H2O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are ΔArg111, ΔThr145, ΔSer177, ΔGlu240, ΔGlu374, and ΔTyr375. The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed to compensate for the electron density donated to oxygen upon the elimination of H2O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of COb towards Fed, which leads to enzyme inhibition. Nevertheless, enzyme reactivation can be achieved by means of appropriate amino acid substitutions

A Regularized and Renormalized Electrostatic Coupling Hamiltonian for hybrid Quantum-Mechanical–Molecular-Mechanical Calculations

We describe a regularized and renormalized electrostatic coupling Hamiltonian for hybrid quantum-mechanical (QM)–molecular-mechanical (MM) calculations. To remedy the nonphysical QM/MM Coulomb interaction at short distances arising from a point electrostatic potential (ESP) charge of the MM atom and also to accommodate the effect of polarized MM atom in the coupling Hamiltonian, we propose a partial-wave expansion of the ESP charge and describe the effect of a s-wave expansion, extended over the covalent radius rc, of the MM atom. The resulting potential describes that, at short distances, large scale cancellation of Coulomb interaction arises intrinsically from the localized expansion of the MM point charge and the potential self-consistently reduces to 1∕rc at zero distance providing a renormalization to the Coulomb energy near interatomic separations. Employing this renormalized Hamiltonian, we developed an interface between the Car-Parrinello molecular-dynamics program and the classical molecular-dynamics simulation program Groningen machine for chemical simulations. With this hybrid code we performed QM/MM calculations on water dimer, imidazole carbon monoxide (CO) complex, and imidazole-heme-CO complex with CO interacting with another imidazole. The QM/MM results are in excellent agreement with experimental data for the geometry of these complexes and other computational data found in literature

Residue Mutations in [Fe-Fe]-Hydrogenase Impedes O 2 Binding: A QM/MM Investigation

[Fe-Fe]-hydrogenases are enzymes that reversibly catalyze the reaction of protons and electrons to molecular hydrogen, which occurs in anaerobic media. In living systems, [Fe-Fe]-hydrogenases are mostly used for H(2) production. The [Fe-Fe]-hydrogenase H-cluster is the active site, which contains two iron atoms. The latest theoretical investigations1,2 advocate that the structure of di-iron air inhibited species are either Fe(p) (II)-Fe(d) (II)-O-H(-), or Fe(p) (II)-Fe(d) (II)-O-O-H, thus O(2) has to be prevented from binding to Fe(d) in all di-iron subcluster oxidation states in order to retain a catalytically active enzyme. By performing residue mutations on [Fe-Fe]-hydrogenases, we were able to weaken O(2) binding to distal iron (Fe(d)) of Desulfovibrio desulfuricans hydrogenase (DdH). Individual residue deletions were carried out in the 8 A apoenzyme layer radial outward from Fe(d) to determine what residue substitutions should be made to weaken O(2) binding. Residue deletions and substitutions were performed for three di-iron subcluster oxidation states, Fe(p) (II)-Fe(d) (II), Fe(p) (II)-Fe(d) (I), and Fe(p) (I)-Fe(d) (I) of [Fe-Fe]-hydrogenase. Two deletions (DeltaThr(152) and DeltaSer(202)) were found most effective in weakening O(2) binding to Fe(d) in Fe(p) (II)-Fe(d) (I) hydrogenase (DeltaG(QM/MM) = +5.4 kcal/mol). An increase in Gibbs\u27 energy (+2.2 kcal/mol and +4.4 kcal/mol) has also been found for Fe(p) (II)-Fe(d) (II), and Fe(p) (I)-Fe(d) (I) hydrogenase respectively. pi-backdonation considerations for frontier molecular orbital and geometrical analysis corroborate the Gibbs\u27s energy results

Inactivation of [Fe-Fe]-Hydrogenase by O2. Thermodynamics and Frontier Molecular Orbitals Analyses

The oxidation of H-cluster in gas phase, and in aqueous enzyme phase, has been investigated by means of quantum mechanics (QM) and combined quantum mechanics–molecular mechanics (QM/MM). Several potential reaction pathways (in the above-mentioned chemical environments) have been studied, wherein only the aqueous enzyme phase has been found to lead to an inhibited hydroxylated cluster. Specifically, the inhibitory process occurs at the distal iron (Fed) of the catalytic H-cluster (which isalso the atom involved in H2 synthesis). The processes involved in the H-cluster oxidative pathways are O2 binding, e− transfer, protonation, and H2O removal. We found that oxygen binding is nonspontaneous in gas phase, and spontaneous for aqueous enzyme phase where both Fe atoms have oxidation state II; however, it is spontaneous for the partially oxidized and reduced clusters in both phases. Hence, in the protein environment the hydroxylated H-cluster is obtained by means of completely exergonic reaction pathway starting with proton transfer. A unifying endeavor has been carried out for the purpose of understanding the thermodynamic results vis-à-vis several other performed electronic structural methods, such as frontier molecular orbitals (FMO), natural bond orbital partial charges (NBO), and H-cluster geometrical analysis. An interesting result of the FMO examination (for gas phase) is that an e− is transferred to LUMOα rather than to SOMOβ, which is unexpected because SOMOβ usually resides in a lower energy rather than LUMOα for open-shell clusters

Aminoacyl-tRNA Synthetases of the Multi-tRNA Synthetase Complex and Their Role in Tumorigenesis

In mammalian cells, 20 aminoacyl-tRNA synthetases (AARS) catalyze the ligation of amino acids to their cognate tRNAs to generate aminoacylated-tRNAs. In higher eukaryotes, 9 of the 20 AARSs, along with 3 auxiliary proteins, join to form the cytoplasmic multi-tRNA synthetase complex (MSC). The complex is absent in prokaryotes, but evolutionary expansion of MSC constituents, primarily by addition of novel interacting domains, facilitates formation of subcomplexes that join to establish the holo-MSC. In some cases, environmental cues direct the release of constituents from the MSC which enables the execution of non-canonical, i.e., moonlighting , functions distinct from their essential activities in protein translation. These activities are generally beneficial, but can also be deleterious to the cell. Elucidation of the non-canonical activities of several AARSs residing in the MSC suggest they are potential therapeutic targets for cancer, as well as metabolic and neurologic diseases. Here, we describe the role of MSC-resident AARSs in cancer progression, and the factors that regulate their release from the MSC. Also, we highlight recent developments in therapeutic modalities that target MSC AARSs for cancer prevention and treatment

Combining Orthogonal tRNA/synthatase Pair and Amber Codon Suppression to Genetically Encode Oxidative Damage in High Density Lipoproteins

Apolipoprotein A-I (apoA-I) is the main protein constituent of high density lipoprotein (HDL - the “good cholesterol”). Oxidatively damaged apoA-I has been isolated from circulating plasma and atherosclerosis plaque with the amino acid residue tryptophan 72 (W72) of apoA-I identified as a primary oxidation site. ApoA-I designed to include specific oxidized amino acids can be used to further investigate the role of site-specific oxidative damage in atherosclerosis. Genetic encoding of oxidized amino acids through orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs offers a reliable method for producing site-specific oxidized proteins. Our project involves the generation of Saccharomyces tryptophan-RS mutants for recognition of oxidized tryptophan (ox-W) but not naturally occurring tryptophan. To study the role of oxidative damage on HDL function we need oxidized proteins that mimic the oxidatively damaged protein observed in vivo. Thus, our goal was to produce site-specific oxidized apoA-I (ox-W72) for incorporation into reconstituted nascent HDL. The two aims we hope to achieve within this project are 1) to provide targeted mutations that increase the specificity and affinity of aaRS towards 2-hydroxy-W-apoA-I, as well as 2) express and confirm the presence of ox-W72 in modified apoA-I.https://engagedscholarship.csuohio.edu/u_poster_2013/1004/thumbnail.jp

Numerical Simulation and Graphical Analysis of In Vitro Benign Tumor Growth: Application of Single-Particle State Bosonic Matter Equation with Length Scaling

We describe the application of a non-linear single-particle state bosonic condensate equation to simulate multicellular tumor growth by treating it as a coupling of two classical wave equations with real components. With one component representing the amplitude of the cells in their volume growth phase and the other representing the amplitude of the cells in their proliferation or mitosis phase, the two components of the coupled equation feed each other during the time evolution and are coupled together through diffusion and other linear and non-linear terms. The features of quiescent and necrotic cells, which result from poor nutrient diffusion into a tumor, have been found to correspond quite well to experimental data when they are modeled as depending on higher cell density. Classical hallmarks of benign tumor growth, such as the initial rapid growth, followed by a dramatic collapse in the proliferating cell count and a strong re-growth thereafter appear quite encouragingly in the theoretical results. A tool for graphical analysis of the tumor simulation results has been developed to provide morphological information about tumors at various growth stages. The model and the graphical analysis can be extended further to create an effective tool to predict/monitor tumor growth