A Targeted Deep Sequencing Method to Quantify Endogenous Retrovirus Gag Sequence Variants and Open Reading Frames Expressed in Nonobese Diabetic Mice.

Endogenous retroviruses (ERVs) are involved in autoimmune diseases such as type 1 diabetes (T1D). ERV gene products homologous to murine leukemia retroviruses are expressed in the pancreatic islets of NOD mice, a model of T1D. One ERV gene, Gag, with partial or complete open reading frames (ORFs), is detected in the islets, and it contains many sequence variants. An amplicon deep sequencing analysis was established by targeting a conserved region within the Gag gene to compare NOD with T1D-resistant mice or different ages of prediabetic NOD mice. We observed that the numbers of different Gag variants and ORFs are linked to T1D susceptibility. More importantly, these numbers change during the course of diabetes development and can be quantified to calculate the levels of disease progression. Sequence alignment analysis led to identification of additional markers, including nucleotide mismatching and amino acid consensus at specific positions that can distinguish the early and late stages, before diabetes onset. Therefore, the expression of sequence variants and ORFs of ERV genes, particularly Gag, can be quantified as biomarkers to estimate T1D susceptibility and disease progression.

DFT Calculations for Mössbauer Properties on Dinuclear Center Models of the Resting Oxidized Cytochrome c Oxidase.

Mössbauer isomer shift and quadrupole splitting properties have been calculated using the OLYP-D3(BJ) density functional method on previously obtained (W.-G. Han Du, et al., Inorg Chem. 2020, 59, 8906-8915) geometry optimized Fea33+ -H2 O-CuB2+ dinuclear center (DNC) clusters of the resting oxidized (O state) "as-isolated" cytochrome c oxidase (CcO). The calculated results are highly consistent with the available experimental observations. The calculations have also shown that the structural heterogeneities of the O state DNCs implicated by the Mössbauer experiments are likely consequences of various factors, particularly the variable positions of the central H2 O molecule between the Fea33+ and CuB2+ sites in different DNCs, whether or not this central H2 O molecule has H-bonding interaction with another H2 O molecule, the different spin states having similar energies for the Fea33+ sites, and whether the Fea33+ and CuB2+ sites are ferromagnetically or antiferromagnetically spin-coupled.

Sulfur [18F]Fluoride Exchange Click Chemistry Enabled Ultrafast Late-Stage Radiosynthesis.

The lack of efficient [18F]fluorination processes and target-specific organofluorine chemotypes remains the major challenge of fluorine-18 positron emission tomography (PET). We report here an ultrafast isotopic exchange method for the radiosynthesis of novel PET agent aryl [18F]fluorosulfate enabled by the emerging sulfur fluoride exchange (SuFEx) click chemistry. The method has been applied to the fully automated 18F-radiolabeling of 25 structurally and functionally diverse aryl fluorosulfates with excellent radiochemical yield (83-100%, median 98%) and high molar activity (280 GBq µmol-1) at room temperature in 30 s. The purification of radiotracers requires no time-consuming HPLC but rather a simple cartridge filtration. We further demonstrate the imaging application of a rationally designed poly(ADP-ribose) polymerase 1 (PARP1)-targeting aryl [18F]fluorosulfate by probing subcutaneous tumors in vivo.

A Water Molecule Residing in the Fea33+···CuB2+ Dinuclear Center of the Resting Oxidized as-Isolated Cytochrome c Oxidase: A Density Functional Study.

Although the dinuclear center (DNC) of the resting oxidized "as-isolated" cytochrome c oxidase (CcO) is not a catalytically active state, its detailed structure, especially the nature of the bridging species between the Fea33+ and CuB2+ metal sites, is still both relevant and unsolved. Recent crystallographic work has shown an extended electron density for a peroxide type dioxygen species (O1-O2) bridging the Fea3 and CuB centers. In this paper, our density functional theory (DFT) calculations show that the observed peroxide type electron density between the two metal centers is most likely a mistaken analysis due to overlap of the electron density of a water molecule located at different positions between apparent O1 and O2 sites in DNCs of different CcO molecules with almost the same energy. Because the diffraction pattern and the resulting electron density map represent the effective long-range order averaged over many molecules and unit cells in the X-ray structure, this averaging can lead to an apparent observed superposition of different water positions between the Fea33+ and CuB2+ metal sites.

Coupled transport of electrons and protons in a bacterial cytochrome c oxidase-DFT calculated properties compared to structures and spectroscopies.

After a general introduction to the features and mechanisms of cytochrome c oxidases (CcOs) in mitochondria and aerobic bacteria, we present DFT calculated physical and spectroscopic properties for the catalytic reaction cycle compared with experimental observations in bacterial ba3 type CcO, also with comparisons/contrasts to aa3 type CcOs. The Dinuclear Complex (DNC) is the active catalytic reaction center, containing a heme a3 Fe center and a near lying Cu center (called CuB) where by successive reduction and protonation, molecular O2 is transformed to two H2O molecules, and protons are pumped from an inner region across the membrane to an outer region by transit through the CcO integral membrane protein. Structures, energies and vibrational frequencies for Fe-O and O-O modes are calculated by DFT over the catalytic cycle. The calculated DFT frequencies in the DNC of CcO are compared with measured frequencies from Resonance Raman spectroscopy to clarify the composition, geometry, and electronic structures of different intermediates through the reaction cycle, and to trace reaction pathways. X-ray structures of the resting oxidized state are analyzed with reference to the known experimental reaction chemistry and using DFT calculated structures in fitting observed electron density maps. Our calculations lead to a new proposed reaction pathway for coupling the PR → F → OH (ferryl-oxo → ferric-hydroxo) pathway to proton pumping by a water shift mechanism. Through this arc of the catalytic cycle, major shifts in pKa's of the special tyrosine and a histidine near the upper water pool activate proton transfer. Additional mechanisms for proton pumping are explored, and the role of the CuB+ (cuprous state) in controlling access to the dinuclear reaction site is proposed.

DFT Fea3-O/O-O Vibrational Frequency Calculations over Catalytic Reaction Cycle States in the Dinuclear Center of Cytochrome c Oxidase.

Density functional vibrational frequency calculations have been performed on eight geometry optimized cytochrome c oxidase (CcO) dinuclear center (DNC) reaction cycle intermediates and on the oxymyoglobin (oxyMb) active site. The calculated Fe-O and O-O stretching modes and their frequency shifts along the reaction cycle have been compared with the available resonance Raman (rR) measurements. The calculations support the proposal that in state A[Fea33+-O2-•···CuB+] of CcO, O2 binds with Fea32+ in a similar bent end-on geometry to that in oxyMb. The calculations show that the observed 20 cm-1 shift of the Fea3-O stretching mode from the PR to F state is caused by the protonation of the OH- ligand on CuB2+ (PR[Fea34+═O2-···HO--CuB2+] → F[Fea34+═O2-···H2O-CuB2+]), and that the H2O ligand is still on the CuB2+ site in the rR identified F[Fea34+═O2-···H2O-CuB2+] state. Further, the observed rR band at 356 cm-1 between states PR and F is likely an O-Fea3-porphyrin bending mode. The observed 450 cm-1 low Fea3-O frequency mode for the OH active oxidized state has been reproduced by our calculations on a nearly symmetrically bridged Fea33+-OH-CuB2+ structure with a relatively long Fea3-O distance near 2 Å. Based on Badger's rule, the calculated Fea3-O distances correlate well with the calculated νFe-O-2/3 (νFe-O is the Fea3-O stretching frequency) with correlation coefficient R = 0.973.

A Water Dimer Shift Activates a Proton Pumping Pathway in the PR → F Transition of ba3 Cytochrome c Oxidase.

Broken-symmetry density functional calculations have been performed on the [Fea34+,CuB2+] state of the dinuclear center (DNC) for the PR → F part of the catalytic cycle of ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt), using the OLYP-D3-BJ functional. The calculations show that the movement of the H2O molecules in the DNC affects the pKa values of the residue side chains of Tyr237 and His376+, which are crucial for proton transfer/pumping in ba3 CcO from Tt. The calculated lowest energy structure of the DNC in the [Fea34+,CuB2+] state (state F) is of the form Fea34+═O2-···CuB2+, in which the H2O ligand that resulted from protonation of the OH- ligand in the PR state is dissociated from the CuB2+ site. The calculated Fea34+═O2- distance in F (1.68 Å) is 0.03 Å longer than that in PR (1.65 Å), which can explain the different Fea34+═O2- stretching modes in P (804 cm-1) and F (785 cm-1) identified by resonance Raman experiments. In this F state, the CuB2+···O2- (ferryl-oxygen) distance is only around 2.4 Å. Hence, the subsequent OH state [Fea33+-OH--CuB2+] with a µ-hydroxo bridge can be easily formed, as shown by our calculations.

Water exit pathways and proton pumping mechanism in B-type cytochrome c oxidase from molecular dynamics simulations.

Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. While proton uptake channels as well as water exit channels have been identified for A-type CcOs, the means by which water and protons exit B-type CcOs remain unclear. In this work, we investigate potential mechanisms for proton transport above the dinuclear center (DNC) in ba3-type CcO of Thermus thermophilus. Using long-time scale, all-atom molecular dynamics (MD) simulations for several relevant protonation states, we identify a potential mechanism for proton transport that involves propionate A of the active site heme a3 and residues Asp372, His376 and Glu126(II), with residue His376 acting as the proton-loading site. The proposed proton transport process involves a rotation of residue His376 and is in line with experimental findings. We also demonstrate how the strength of the salt bridge between residues Arg225 and Asp287 depends on the protonation state and that this salt bridge is unlikely to act as a simple electrostatic gate that prevents proton backflow. We identify two water exit pathways that connect the water pool above the DNC to the outer P-side of the membrane, which can potentially also act as proton exit transport pathways. Importantly, these water exit pathways can be blocked by narrowing the entrance channel between residues Gln151(II) and Arg449/Arg450 or by obstructing the entrance through a conformational change of residue Tyr136, respectively, both of which seem to be affected by protonation of residue His376.

A broken-symmetry density functional study of structures, energies, and protonation states along the catalytic O-O bond cleavage pathway in ba3 cytochrome c oxidase from Thermus thermophilus.

Broken-symmetry density functional calculations have been performed on the [Fea3, CuB] dinuclear center (DNC) of ba3 cytochrome c oxidase from Thermus thermophilus in the states of [Fea3(3+)-(HO2)(-)-CuB(2+), Tyr237(-)] and [Fea3(4+)[double bond, length as m-dash]O(2-), OH(-)-CuB(2+), Tyr237˙], using both PW91-D3 and OLYP-D3 functionals. Tyr237 is a special tyrosine cross-linked to His233, a ligand of CuB. The calculations have shown that the DNC in these states strongly favors the protonation of His376, which is above propionate-A, but not of the carboxylate group of propionate-A. The energies of the structures obtained by constrained geometry optimizations along the O-O bond cleavage pathway between [Fea3(3+)-(O-OH)(-)-CuB(2+), Tyr237(-)] and [Fea3(4+)[double bond, length as m-dash]O(2-)HO(-)-CuB(2+), Tyr237˙] have also been calculated. The transition of [Fea3(3+)-(O-OH)(-)-CuB(2+), Tyr237(-)] → [Fea3(4+)[double bond, length as m-dash]O(2-)HO(-)-CuB(2+), Tyr237˙] shows a very small barrier, which is less than 3.0/2.0 kcal mol(-1) in PW91-D3/OLYP-D3 calculations. The protonation state of His376 does not affect this O-O cleavage barrier. The rate limiting step of the transition from state A (in which O2 binds to Fea3(2+)) to state PM ([Fea3(4+)[double bond, length as m-dash]O(2-), OH(-)-CuB(2+), Tyr237˙], where the O-O bond is cleaved) in the catalytic cycle is, therefore, the proton transfer originating from Tyr237 to O-O to form the hydroperoxo [Fea3(3+)-(O-OH)(-)-CuB(2+), Tyr237(-)] state. The importance of His376 in proton uptake and the function of propionate-A/neutral-Asp372 as a gate to prevent the proton from back-flowing to the DNC are also shown.

A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans.

Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission maximum of probe 4 in comparison to its nonfluorosulfated analogue. We demonstrate that probe 4 is dark in aqueous buffers, whereas the TTR·4 complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR·4 fluorescence in macrophage-like coelomocytes. An analogue of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analogue suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.

Broken Symmetry DFT Calculations/Analysis for Oxidized and Reduced Dinuclear Center in Cytochrome c Oxidase: Relating Structures, Protonation States, Energies, and Mössbauer Properties in ba3 Thermus thermophilus.

The Fea3(3+)···CuB(2+) dinuclear center (DNC) structure of the as-isolated oxidized ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced ba3 CcO from Tt are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcO's were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fea3(2+)···CuB(+) DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba3 CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779-5783) revealed multiple (57)Fea3(2+) and (57)Fea3(3+) components. Moreover, one of the (57)Fea3(3+) components observed at 4.2 K transformed from a proposed "low-spin" state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin (57)Fea3(3+) component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the (57)Fea3(3+) components, and how the coordination and spin states of the Fea3(3+/2+) and Cu(2+/1+) sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of ba3 CcO from Tt. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the CuB(+) site, and they may become potential proton loading sites during the catalytic cycle.

Linking chemical electron-proton transfer to proton pumping in cytochrome c oxidase: broken-symmetry DFT exploration of intermediates along the catalytic reaction pathway of the iron-copper dinuclear complex.

After a summary of the problem of coupling electron and proton transfer to proton pumping in cytochrome c oxidase, we present the results of our earlier and recent density functional theory calculations for the dinuclear Fe-a3-CuB reaction center in this enzyme. A specific catalytic reaction wheel diagram is constructed from the calculations, based on the structures and relative energies of the intermediate states of the reaction cycle. A larger family of tautomers/protonation states is generated compared to our earlier work, and a new lowest-energy pathway is proposed. The entire reaction cycle is calculated for the new smaller model (about 185-190 atoms), and two selected arcs of the wheel are chosen for calculations using a larger model (about 205 atoms). We compare the structural and redox energetics and protonation calculations with available experimental data. The reaction cycle map that we have built is positioned for further improvement and testing against experiment.

Density functional study for the bridged dinuclear center based on a high-resolution X-ray crystal structure of ba3 cytochrome c oxidase from Thermus thermophilus.

Strong electron density for a peroxide type dioxygen species bridging the Fea3 and CuB dinuclear center (DNC) was observed in the high-resolution (1.8 Å) X-ray crystal structures (PDB entries 3S8G and 3S8F) of ba3 cytochrome c oxidase (CcO) from Thermus thermophilus. The crystals represent the as-isolated X-ray photoreduced CcO structures. The bridging peroxide was proposed to arise from the recombination of two radiation-produced HO(•) radicals formed either very near to or even in the space between the two metals of the DNC. It is unclear whether this peroxide species is in the O2(2-), O2(•)(-), HO2(-), or the H2O2 form and what is the detailed electronic structure and binding geometry including the DNC. In order to answer what form of this dioxygen species was observed in the DNC of the 1.8 Å X-ray CcO crystal structure (3S8G), we have applied broken-symmetry density functional theory (BS-DFT) geometric and energetic calculations (using OLYP potential) on large DNC cluster models with different Fea3-CuB oxidation and spin states and with O2(2-), O2(•)(-), HO2(-), or H2O2 in the bridging position. By comparing the DFT optimized geometries with the X-ray crystal structure (3S8G), we propose that the bridging peroxide is HO2(-). The X-ray crystal structure is likely to represent the superposition of the Fea3(2+)-(HO2(-))-CuB(+) DNC's in different states (Fe(2+) in low spin (LS), intermediate spin (IS), or high spin (HS)) with the majority species having the proton of the HO2(-) residing on the oxygen atom (O1) which is closer to the Fea3(2+) site in the Fea3(2+)-(HO-O)(-)-CuB(+) conformation. Our calculations show that the side chain of Tyr237 is likely trapped in the deprotonated Tyr237(-) anion form in the 3S8G X-ray crystal structure.

Reaction pathways, proton transfer, and proton pumping in ba3 class cytochrome c oxidase: perspectives from DFT quantum chemistry and molecular dynamics.

After drawing comparisons between the reaction pathways of cytochrome c oxidase (CcO, Complex 4) and the preceding complex cytochrome bc1 (Complex 3), both being proton pumping complexes along the electron transport chain, we provide an analysis of the reaction pathways in bacterial ba3 class CcO, comparing spectroscopic results and kinetics observations with results from DFT calculations. For an important arc of the catalytic cycle in CcO, we can trace the energy pathways for the chemical protons and show how these pathways drive proton pumping of the vectorial protons. We then explore the proton loading network above the Fe heme a3-CuB catalytic center, showing how protons are loaded in and then released by combining DFT-based reaction energies with molecular dynamics simulations over states of that cycle. We also propose some additional reaction pathways for the chemical and vector protons based on our recent work with spectroscopic support.

Data for molecular dynamics simulations of B-type cytochrome c oxidase with the Amber force field.

Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. This article presents parameters for the cofactors of ba3-type CcO that are compatible with the all-atom Amber ff12SB and ff14SB force fields. Specifically, parameters were developed for the CuA pair, heme b, and the dinuclear center that consists of heme a3 and CuB bridged by a hydroperoxo group. The data includes geometries in XYZ coordinate format for cluster models that were employed to compute proton transfer energies and derive bond parameters and point charges for the force field using density functional theory. Also included are the final parameter files that can be employed with the Amber leap program to generate input files for molecular dynamics simulations with the Amber software package. Based on the high resolution (1.8 Å) X-ray crystal structure of the ba3-type CcO from Thermus thermophilus (Protein Data Bank ID number PDB: 3S8F), we built a model that is embedded in a POPC lipid bilayer membrane and solvated with TIP3P water molecules and counterions. We provide PDB data files of the initial model and the equilibrated model that can be used for further studies.

EXAFS simulation refinement based on broken-symmetry DFT geometries for the Mn(IV)-Fe(III) center of class I RNR from Chlamydia trachomatis.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides necessary for DNA biosynthesis. Unlike the conventional class Ia RNRs which use a diiron cofactor in their subunit R2, the active site of the RNR-R2 from Chlamydia trachomatis (Ct) contains a Mn/Fe cofactor. The detailed structure of the Mn/Fe core has yet to be established. In this paper we evaluate six different structural models of the Ct RNR active site in the Mn(iv)/Fe(iii) state by using Mössbauer parameter calculations and simulations of Mn/Fe extended X-ray absorption fine structure (EXAFS) spectroscopy, and we identify a structure similar to a previously proposed DFT-optimized model that shows quantitative agreement with both EXAFS and Mössbauer spectroscopic data.