In silico modeling of pH-optimum of protein-protein binding

Protein-protein association is a pH-dependent process and thus the binding affinity depends on the local pH. In vivo the association occurs in a particular cellular compartment, where the individual monomers are supposed to meet and form a complex. Since the monomers and the complex exist in the same micro environment, it is plausible that they coevolved toward its properties, in particular, toward the characteristic subcellular pH. Here we show that the pH at which the monomers are most stable (pH-optimum) or the pH at which stability is almost pH-independent (pH-flat) of monomers are correlated with the pH-optimum of maximal affinity (pH-optimum of binding) or pH interval at which affinity is almost pH-independent (pH-flat of binding) of the complexes made of the corresponding monomers. The analysis of interfacial properties of protein complexes demonstrates that pH-dependent properties can be roughly estimated using the interface charge alone. In addition, we introduce a parameter beta, proportional to the square root of the absolute product of the net charges of monomers, and show that protein complexes characterized with small or very large beta tend to have neutral pH-optimum. Further more, protein complexes made of monomers carrying the same polarity net charge at neutral pH have either very low or very high pH-optimum of binding. These findings are used to propose empirical rule for predicting pH-optimum of binding provided that the amino acid compositions of the corresponding monomers are available.     

Structural perturbation of human hemoglobin on glutathionylation probed by hydrogen-deuterium exchange and MALDI mass spectrometry

Glutathionyl hemoglobin, an example of post-translationally modified hemoglobin, has been studied as a marker of oxidative stress in various diseased conditions. Compared to normal hemoglobin, glutathionyl hemoglobin has been found to have increased oxygen affinity and reduced cooperativity. However, detailed information concerning the structural perturbation of hemoglobin associated with glutathionylation is lacking. In the present study, we report structural changes associated with glutathionylation of deoxyhemoglobin by hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry. We analyzed isotope exchange kinetics of backbone amide hydrogen of eleven peptic peptides in the deoxy state of both hemoglobin and glutathionyl hemoglobin molecules. Analysis of the deuterium incorporation kinetics for both molecules showed structural changes associated with the following peptides: α34-46, α1-29, β32-41, β86-102, β115-129, and β130-146. H/D exchange experiments suggest that glutathionylation of hemoglobin results in a change in conformation located at the above-mentioned regions of the hemoglobin molecule. MALDI mass spectrometry based H/D exchange experiment might be a simple way of monitoring structural changes associated with post-translational modification of protein.     

RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Identifying polyglutamine protein species in situ that best predict neurodegeneration

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.     

Attenuation of oxidative stress by allylpyrocatechol in synovial cellular infiltrate of patients with Rheumatoid Arthritis

Free radicals are involved in the pathogenesis of Rheumatoid arthritis, a systemic autoimmune disorder characterized by unchecked synovial inflammation. Allylpyrocatechol, a phytoconstituent of Piper betle leaves, has potent anti-inflammatory activity and this study evaluated its anti-oxidant effect on the synovial infiltrate of patients with Rheumatoid arthritis. The ex vivo effect of allylpyrocatechol upon generation of reactive oxygen species in neutrophils, macrophages and lymphocytes was measured by flow cytometry using dichlorodihydrofluorescein diacetate, wherein it significantly decreased basal levels as also scavenged phorbol myristate acetate generated reactive oxygen species. Furthermore, its effect on generation of superoxide and hydroxyl radicals produced within infiltrated neutrophils was measured by cytochrome c and deoxyribose assay, respectively. Allylpyrocatechol significantly scavenged superoxide and hydroxyl radicals in infiltrated neutrophils. The effect of allylpyrocatechol on nitric oxide was measured in macrophages using 4,5-diaminofluorescein diacetate by flow cytometry wherein it decreased production of nitric oxide in infiltrated macrophages, which correlated with its in vitro nitric oxide scavenging activity. Taken together, this ex vivo study has established that allylpyrocatechol has potent scavenging activity and could be considered as an add-on therapy in the treatment of inflammation-associated disorders like Rheumatoid Arthritis.     

Sensitive single-molecule protein quantification and protein complex detection in a microarray format

Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here, we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and four orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53-MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.     

Measuring the persistence length of MCF7 cell microtubules in vitro

The dynamic and mechanical properties of mammalian neural microtubules have been widely studied; however, similar knowledge about these properties is limited for non-neural microtubules, which, unlike neural microtubules, consist of different β-tubulin isotypes. In this study, we report, for the first time, an estimated value for the persistence length of a single non-neural microtubule polymerized from purified tubulin from human breast cancer cell lines (MCF7 tubulin). The method of measurement is based on an analysis of the local curvature of a microtubule as a result of thermal fluctuations. In parallel, we measured the persistence length of a single bovine brain microtubule under similar conditions. The results of our measurements indicate a higher value for the persistence length of MCF7 microtubules in vitro as compared to the persistence length of a neural microtubule. The difference can be associated with different β-tubulin isotypes in the structure of MCF7 microtubules.     

The interplay of O-H?O hydrogen bonding in the generation of three new supramolecular complexes of Cu, Ni and Co: Syntheses, characterization and structural aspects

Three new coordination complexes, [Cu(L¹)(H₂O)] (1), [Ni(L²)₂]·CH₃CN (2) and [Co(HL³)(L³)] (3) [where H₂L¹, N,N′-bis(3-methoxysalicylidenimino)-1,3-diamino-propane; HL², 2-((E)-(1,3-dihydroxy-2-methylpropan-2-ylimino)methyl)phenol; H₂L³, 2-((E)-(2-hydroxyethylimino)methyl)-4-bromophenol] have been synthesized and systematically characterized by elemental analyses, FT-IR, electronic spectroscopy, cyclic voltammetry and thermogravimetric analyses. Single crystal X-ray diffraction studies confirm that the metal center in complex 1 has distorted square-pyramidal geometry while it is distorted octahedral in the other two complexes. In all the complexes O-H?O hydrogen bondings assemble the molecular units leading to ordered supramolecular architectures. While both complexes 1 and 2 form infinite one-dimensional arrays through the self organisation of hydrogen bonded ring motifs, complex 3 is a unique star-shaped cyclic hexamer generated through intermolecular hydrogen bonding.