Stable Dimer Research Articles

Uncovering allostery and regulation in SORCIN through molecular dynamics simulations

Soluble resistance-related calcium-binding protein or Sorcin is an allosteric, calcium-binding Penta-EF hand (PEF) family protein implicated in multi-drug resistant cancers. Sorcin is known to bind chemotherapeutic molecules such as Doxorubicin. This study uses in-silico molecular dynamics simulations to explore the dynamics and allosteric behavior of Sorcin in the context of Ca2+ uptake and Doxorubicin binding. The results show that Ca2+ binding induces large, but reversible conformational changes in the Sorcin structure which manifest as rigid body reorientations that preserve the local secondary structure. A reciprocal allosteric handshake centered around the EF5 hand is found to be key in Sorcin dimer formation and stabilization. Binding of Doxorubicin results in rearrangement of allosteric communities which disrupts long-range allosteric information transfer from the N-terminal domain to the middle lobe. However, this binding does not result in secondary structure destabilization. Sorcin does not appear to have a distinct Ca2+ activated mode of Doxorubicin binding. Communicated by Ramaswamy H. Sarma

Achieving Simplified and Tunable Flexibility in Carborane-Based Emitters for Quantitative Vapochromic VOC Sensing.

Photoluminescence (PL) sensing of volatile organic compounds (VOCs) represents a convenient and economic detection method toward air pollutants. However, tetraphenylethylene (TPE)-based and recent carborane (Cb)-based sensors retained multiple sites that are responsive to VOC stimulation, making quantitative PL sensing rather challenging. Rendering the simplified and tunable flexibility in the PL sensors is key to achieve the quantitative target. In this work, we proposed a dimeric model of Cb-based emitters to deal with flexibility. Three emissive dibenzothiophene (DBT)-alkynylated carboranes (Cb-1/2/3) were designed and synthesized. Among them, Cb-3 contributed green and green-yellow emission in the crystals, as well as yellow and orange emission in the VOC-incorporated films, together unfolding its vapochromic properties. Crystallographic studies revealed that Cb-3 molecules were invariably dimerized in an interlocked fashion and the redshift in PL was caused by the successive through-space conjugation of DBT moieties. Theoretical calculations verified the thermodynamics stability of Cb-3 dimers and suggested that DBT could individually rotate different angles under the simulation of VOCs. Based on the above findings, we introduced DBT-alkynylated carboranes to detect the VOCs and established linear relationships between the photon energy at the PL maxima and the concentrations of benzene and tetrahydrofuran (THF) vapors. Aside from the successful implementation of quantitative vapochromic sensing, the fast response (6 s) and recovery (3∼5 s), as well as the good reusability, were also evidenced in the sensing of THF vapors.

Abstract 1480: ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for the acquisition of malignant cells, which causes epithelial cells with specialized cell-cell contacts to gain mesenchymal migratory capacity. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated with poor clinical outcomes of cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a calculated molecular weight of 125kDa; however, numerous groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutant-expressing NSCLC cell lines. We hypothesize that ZEB1 dimerization is regulated by K811 acetylation to promote protein stability and lung adenocarcinoma metastasis. We determined that the K811R ZEB1 (125 kDa) has a shorter half-life wild-type and high ubiquitination rate than (WT) ZEB1 and K811Q ZEB1 (~225 kDa), suggesting that disruption of ZEB1 acetylation hinders its dimerization and protein stability. Further, ZEB1 dimer recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that unlike K811R ZEB1, WT ZEB1 and K811Q ZEB1 downregulate the expression of gene sets involved in tight junction, apical junction, and cell-cell junction formation. We also identified six novel ZEB1 acetylation-dependent gene candidates that function in signaling pathways and epithelial cell maintenance; CBLN3, STAP2, PLEKHG6, LAD1, RNF43, and LLGL2. In vitro 2D migration Boyden chamber, 3D invasion assays, and in vivo studies demonstrated that ZEB1 acetylation promotes the invasive and metastatic potential of the NSCLC cell line. Our findings suggest that K811 acetylation regulates ZEB1 dimerization and stability to promote NuRD interaction and lung adenocarcinoma via EMT. Future studies are focused on assessing changes in gene expression programs and chromatin accessibility that are ZEB1 dimerization dependent and that could contribute to NSCLC metastasis. Citation Format: Mabel Giselle Perez-Oquendo, Roxsan Manshouri, Yanhua Tian, Jared Fradette, Don Gibbons. ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1480.

Crystal Structure and Functional Characterization of an S-Formylglutathione Hydrolase (BuSFGH) from Burkholderiaceae sp.

S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities.

Unusual pKa Values Mediate the Self-Assembly of Spider Dragline Silk Proteins.

Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.

Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson's disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

Numerical study of magneto-optical binding between two dipolar particles under illumination by two counter-propagating waves

Introduction: The formation of a stable magneto plasmonic dimer with THz resonances is theoretically studied for the principal directions of the system. Unlike a recent report, our work provides a complete description of the full photonic coupling for arbitrary magnetic fields as, for instance, unbalanced particle spins.Methods: As an illustration, we consider two small, n-doped InSb nanoparticles under illumination by two counter-propagating plane waves.Results: Remarkably, when an external magnetic field exists, the symmetry in the system is broken, and a resonant radiation pressure for the dimer appears. Similarly, tunable inter-particle forces and spins are exerted on the non-reciprocal dimer. The system is also characterized when the magnetic field is absent. Moreover, we show how the mechanical observables truly characterize the dimer since their resonance dependency contains detailed information about the system.Discussion: Unlike far-field observables like absorption, mechanical magnitudes depend on the system's near-field. In addition, the nature of the particle spins is originally explained by the energy flow's behavior around the dimer. This work constitutes a generalization of any previous approach to optical binding between small nanoparticles. It paves the way for fully controlling optical matter and nano factory designs based on surface plasmon polaritons.

Stereochemistry of the Reactions between Palladacycle Complexes and Primary Alkyl Iodides

As an important elementary step in organometallic chemistry, the alkylation reaction of palladacycle complexes and alkyl halides has attracted much attention in recent years due to their presence as a key step in palladium-catalyzed C–H alkylation reactions. In principle, several alkylation mechanisms, such as the stereoinvertive SN2-type mechanism and the stereoretentive oxidative addition mechanism, can be operative, and mechanistic insights can be obtained from the stereochemical outcomes of these alkylation reactions. Previous stereochemical investigations on the alkylation reaction of palladacycle complexes mainly focused on the use of chiral secondary alkyl halides as stereochemical probes, leaving more synthetically relevant primary alkyl iodides untouched. In this work, deuterium-labeled primary alkyl iodides were selected as a stereochemical probe, and their reaction with C,C- and C,N-type palladacycle complexes, namely, Catellani-type palladacycle intermediates and directing group (DG)-coordinated palladacycle complexes, were investigated both experimentally and computationally to elucidate the alkylation mechanism. We found that the C,C-ligated palladacycle intermediates undergo alkylation through the SN2-type mechanism, while the C,N-ligated 8-aminoquinoline-derived palladacycle complex favors a stereoretentive oxidative addition mechanism. In addition, the 2-phenylpyridine-derived C,N-type palladacycle dimer complex was found to react through the SN2-type mechanism due to its stable dimer structure and the d8–d8 interaction between two palladium atoms.

Study of novel androgen receptor V770 variant in androgen insensitivity syndrome patients reveals the transitional state of the androgen receptor ligand binding domain homodimer.

We report the discovery of the androgen receptor missense mutation V770D, that was found in two sisters suffering from complete androgen insensitivity. Experimental validation of AR V770 variants demonstrated that AR V770D was transcriptionally inactive due to the inability to dimerize and a reduced ligand binding affinity. The more conservative AR V770A variant showed a dimerization defect at low levels of DHT with a partial recovery of the transcriptional activity and of the receptor's ability to dimerize when increasing the DHT levels. With V770 located outside of the proposed LBD dimerization interface of the AR LBD homodimer crystal structure, the effects of the V770A mutation on AR dimerization were unexpected. We therefore explored whether the AR LBD dimerization interface would be better described by an alternative dimerization mode based on available human homodimeric LBD crystal structures of other nuclear receptors. Superposition of the monomeric AR LBD in the homodimeric crystal structures of GR, PR, ER, CAR, TRβ, and HNF-4α showed that the GR-like LBD dimer model was energetically the most stable. Moreover, V770 was a key energy residue in the GR-like LBD dimer while it was not involved in the stabilization of the AR LBD homodimer according to the crystal structure. Additionally, the observation that 4 AIS mutations impacted the stability of the AR LBD dimer while 16 mutations affected the GR-like LBD dimer, suggested that the AR LBD dimer crystal is a snapshot of a breathing AR LBD homodimer that can transition into the GR-like LBD dimer model.

Exceptionally Stable Dimers and Trimers of Au25 Clusters Linked with a Bidentate Dithiol: Synthesis, Structure and Chirality Study

AbstractA bidentate chiral dithiol (diBINAS) is utilised to bridge Au25 nanoclusters to form oligomers. Separation by size allows the isolation of fractions that are stable thanks to the bidentate nature of the linker. The structure of the products is elucidated by small‐angle X‐ray scattering and calculated using density functional theory. Additional structural details are studied by diffusion‐ordered nuclear magnetic resonance spectroscopy, transmission electron microscopy and matrix‐assisted laser desorption/ionization time of flight mass spectrometry. Significant changes in the optical properties are analysed by UV/Vis and fluorescence spectroscopies, with the latter demonstrating a strong emission enhancement. Furthermore, the emergent chiral characteristics are studied by circular dichroism. Due to the geometry constraints of the nanocluster assemblies, diBINAS can be regarded as a templating molecule, taking a step towards the directed self‐assembly of metal clusters.

Exceptionally Stable Dimers and Trimers of Au25 Clusters Linked with a Bidentate Dithiol: Synthesis, Structure and Chirality Study.

A bidentate chiral dithiol (diBINAS) is utilised to bridge Au25 nanoclusters to form oligomers. Separation by size allows the isolation of fractions that are stable thanks to the bidentate nature of the linker. The structure of the products is elucidated by small-angle X-ray scattering and calculated using density functional theory. Additional structural details are studied by diffusion-ordered nuclear magnetic resonance spectroscopy, transmission electron microscopy and matrix-assisted laser desorption/ionization time of flight mass spectrometry. Significant changes in the optical properties are analysed by UV/Vis and fluorescence spectroscopies, with the latter demonstrating a strong emission enhancement. Furthermore, the emergent chiral characteristics are studied by circular dichroism. Due to the geometry constraints of the nanocluster assemblies, diBINAS can be regarded as a templating molecule, taking a step towards the directed self-assembly of metal clusters.

ATP synthase interactome analysis identifies a new subunit l as a modulator of permeability transition pore in yeast

The mitochondrial ATP synthase, an enzyme that synthesizes ATP and is involved in the formation of the mitochondrial mega-channel and permeability transition, is a multi-subunit complex. In S. cerevisiae, the uncharacterized protein Mco10 was previously found to be associated with ATP synthase and referred as a new ‘subunit l’. However, recent cryo-EM structures could not ascertain Mco10 with the enzyme making questionable its role as a structural subunit. The N-terminal part of Mco10 is very similar to k/Atp19 subunit, which along with subunits g/Atp20 and e/Atp21 plays a major role in stabilization of the ATP synthase dimers. In our effort to confidently define the small protein interactome of ATP synthase we found Mco10. We herein investigate the impact of Mco10 on ATP synthase functioning. Biochemical analysis reveal in spite of similarity in sequence and evolutionary lineage, that Mco10 and Atp19 differ significantly in function. The Mco10 is an auxiliary ATP synthase subunit that only functions in permeability transition.

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Unveiling mutation effects on the structural dynamics of the main protease from SARS-CoV-2 with hybrid simulation methods

The main protease of SARS-CoV-2 (called Mpro or 3CLpro) is essential for processing polyproteins encoded by viral RNA. Several Mpro mutations were found in SARS-CoV-2 variants, which are related to higher transmissibility, pathogenicity, and resistance to neutralization antibodies. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. In this study, we used a hybrid simulation method to generate intermediate structures along the six lowest frequency normal modes and sample the conformational space and characterize the structural dynamics and global motions of WT SARS-CoV-2 Mpro and 48 mutations, including mutations found in P.1, B.1.1.7, B.1.351, B.1.525 and B.1.429+B.1.427 variants. We tried to contribute to the elucidation of the effects of mutation in the structural dynamics of SARS-CoV-2 Mpro. A machine learning analysis was performed following the investigation regarding the influence of the K90R, P99L, P108S, and N151D mutations on the dimeric interface assembling of the SARS-CoV-2 Mpro. The parameters allowed the selection of potential structurally stable dimers, which demonstrated that some single surface aa substitutions not located at the dimeric interface (K90R, P99L, P108S, and N151D) are able to induce significant quaternary changes. Furthermore, our results demonstrated, by a Quantum Mechanics method, the influence of SARS-CoV-2 Mpro mutations on the catalytic mechanism, confirming that only one of the chains of the WT and mutant SARS-CoV-2 Mpros are prone to cleave substrates. Finally, it was also possible to identify the aa residue F140 as an important factor related to the increasing enzymatic reactivity of a significant number of SARS-CoV-2 Mpro conformations generated by the normal modes-based simulations.

Structure of the N-terminal coiled-coil domains of the ciliary protein Rpgrip1l

Rpgrip1l is one of the key ciliary proteins located at the transition zone of the primary cilium, an important organelle for cells to sense the outer environment. Mutations in the RPGRIP1L gene are associated with various ciliopathies. Here, we focused on the N-terminal coiled-coil of Rpgrip1l. By comprehensive biochemical and structural characterizations, we demonstrated that the two predicted coiled-coil regions (CC12) located at Rpgrip1l N-terminus each can form a stable parallel dimer. We further showed that overexpression of Rpgrip1l CC12 in NIH/3T3 cells significantly shortened the length of primary cilia, and this effect depended on the dimer formation. In addition, we found that CC12 of the homolog protein Rpgrip1 in mouse and human were significantly different from Rpgrip1l. Finally, we confirmed that some disease-related mutations can alter the dimeric states of CC12 of Rpgrip1l or Rpgrip1, which might explain the pathogenic mechanisms.

Dissipative stabilization of dark quantum dimers via squeezed vacuum

Understanding the mechanism through which an open quantum system exchanges information with an environment is central to the creation and stabilization of quantum states. This theme has been explored recently, with attention mostly focused on system control or environment engineering. Here, we bring these ideas together to describe the many-body dynamics of an extended atomic array coupled to a squeezed vacuum. We show that fluctuations can drive the array into a pure dark state decoupled from the environment. The dark state is obtained for an even number of atoms and consists of maximally entangled atomic pairs, or dimers, that mimic the behavior of the squeezed field. Each pair displays reduced fluctuations in one polarization quadrature and amplified in another. This dissipation-induced stabilization relies on an efficient transfer of correlations between pairs of photons and atoms. It uncovers the mechanism through which squeezed light causes an atomic array to self-organize and illustrates the increasing importance of spatial correlations in modern quantum technologies where many-body effects play a central role.

The influence of pH on the structure and stability of the Grb2 dimer reveals changes in the inter-domain and molecular interaction: Could it be a modulation mechanism?

Cancer cells present an increased replicative potential as a hallmark. The increased replication leads to a higher intracellular pH. Grb2, an adapter protein, is mainly involved in several types of cancers due to its role in signaling pathways responsible for cell growth and proliferation. At pH 7, we observed a more compact structure, as seen by DLS and 1H NMR relaxation experiments, with high cooperativity within domains. On the other hand, we observed an increase in disordered structures at pH 8, with relative independence between domains characterized by higher melting temperatures and enthalpy of unfolding. CD and DLS corroborate with these observations at pH 8, conferring more flexibility among the domains, followed by lower unfolding cooperativity and increased hydrodynamic diameter at higher pH. In addition, 15N-HSQC chemical shift perturbations experiments showed significant differences in the positions of several amino acids spread on the Grb2 structure when pH was changed, which agrees with the previous results. Finally, the molecular dynamic analysis demonstrates that Grb2 presents a movement pattern where both SH3 domains move toward the center of the protein at pH 7. On the contrary, the pattern changes its direction at pH 8, where domains move outside the center of the protein, conferring a more elongated structure at higher pH. So, Grb2 presents significant structural and dynamic changes modulated by pH. If considering the role of Grb2 in cell signaling upstream, these conformational changes could be a critical mechanistic behavior of this protein, preventing/disrupting the stability of the cell signaling pathways related to cancer.

Diminishing oocyte quality with advancing age is associated with deficiency of nitric oxide synthase cofactors, tetrahydrobiopterin, and zinc, in mouse oocytes

To study the implications of decreased zinc and tetrahydrobiopterin (H4B) associated with chronological aging on oocyte quality using a mouse model. H4B and zinc are essential cofactors for nitric oxide synthase (NOS), because they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown that sufficient levels of nitric oxide (NO) are essential for maintaining oocyte quality and activation, and NO levels decrease in the oocyte as a function of age. Thus, it is plausible that zinc and H4B may decrease as a function of age, resulting in NOS dysfunction with subsequent depletion of NO. Additionally, increased production of reactive oxygen species from the monomeric form can further disrupt oocyte quality and NO bioavailability. Experimental laboratory study. Laboratory. B6D2F1 mice. Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from 3 age groups: 8-14 weeks (young breeders [YBs]), 48-52 weeks (retired breeders [RBs]), and 80-84 weeks (old animals [OAs]). Oocytes were scored for ooplasmic/spindle microtubule (MT) morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3 dimension image reconstruction and subjected to an high-performance liquid chromatography assay to measure the concentrations of H4B and its metabolites, as well as the zinc measurement using atomic absorption spectrophotometry. Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increases, with YB having the highest percentage of quality oocytes followed by RB and OA. The high-performance liquid chromatography analysis showed a significant progressive decrease in total H4B in RB and OA (0.00098 picogram (pg)/oocyte and 0.00069 pg/oocyte, respectively) compared with YB (0.00125 pg/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared with YB (8.45 pg/oocyte and 5.82 pg/oocyte vs. 10.05 pg/oocyte, respectively). Age-related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. The resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide instead of NO, which participates in a multitude of reactions contributing to oxidative stress. Therefore, dysfunction of NOS secondary to zinc and H4B loss is a major mechanism involved in reactive oxygen species generation and oocyte quality deterioration related to the chronological age.

Experimental FTIR-MI and Theoretical Studies of Isocyanic Acid Aggregates.

Homoaggregates of isocyanic acid (HNCO) were studied using FTIR spectroscopy combined with a low-temperature matrix isolation technique and quantum chemical calculations. Computationally, the structures of the HNCO dimers and trimers were optimized at the MP2, B3LYPD3 and B2PLYPD3 levels of theory employing the 6-311++G(3df,3pd) basis set. Topological analysis of the electron density (AIM) was used to identify the type of non-covalent interactions in the studied aggregates. Five stable minima were located on the potential energy surface for (HNCO)2, and nine were located on the potential energy surface for (HNCO)3. The most stable dimer (D1) involves a weak, almost linear N-H⋯N hydrogen bond. Other structures are bound by a N-H⋯O hydrogen bond or by O⋯C or N⋯N van der Waals interactions. Similar types of interactions as in (HNCO)2 were found in the case of HNCO trimers. Among nine stable (HNCO)3 structures, five represent cyclic forms. The most stable T1 trimer structure is characterized by a six-membered ring formed by three N-H⋯N hydrogen bonds and representing high symmetry (C3h). The analysis of the HNCO/Ar spectra after deposition indicates that the N-H⋯O hydrogen-bonded dimers are especially prevalent. Upon annealing, HNCO trimers were observed as well. Identification of the experimentally observed species relied on previous experimental data on HNCO complexes as well as computed data on HNCO homoaggregates' vibrational spectra.

The role of protein-protein interactions in Fluc dimerization stability and function.

Functional ion channel assembly often requires subunit oligomerization. Yet the molecular factors that define the stability of ion channel complexes in membranes remain to be delineated. Here we investigate this question by studying the dynamic dimerization reaction of the model system Fluc to map out the molecular and physical forces driving this reaction in membranes. Fluc is dual-topology homodimer, where dimerization confers the highly specific fluoride permeation pathway. Our previous analysis demonstrated that a sodium titratable mutant of Fluc participates in a two-step equilibrium dimerization reaction in membranes. First, membrane-dependent dimerization drives subunits to assemble and form a non-contact lipid-solvated stable dimer intermediate by burying a membrane defect imposed in the monomer. However, the fluoride conducting state requires the protein subunits to come closer and expel the lipids to form the fluoride permeation pathway. High resolution crystal structures of Fluc show numerous protein-protein interactions across the dimerization interface, suggesting these interactions might be involved in stabilizing the close-contact dimer state. Alternatively, these contacts may not play a major role in the overall dimer stability but may be important for conferring function. Here, we will investigate how disruption of these protein-protein interactions affects dimerization stability, channel activity or both. Site-directed mutagenesis was employed following four strategies of disruption: (1) reduction of van der Waals interactions by leucine-to-alanine substitutions; (2) removal of potential hydrogen bonds by threonine-to-valine substitutions; (3) decrease backbone flexibility by glycine-to-alanine mutations of the GxxxG dimerization motif; (4) and introduction of bulky tryptophan residues at the interface. Using single-molecule photobleaching analysis to investigate dimer stability in membranes combined with single channel bilayer recordings, these results will provide a quantitative characterization of the contribution of protein-protein interactions to Fluc dimer stability and activity.