VLDP web server: a powerful geometric tool for analysing protein structures in their environment.

International audienceProtein structures are an ensemble of atoms determined experimentally mostly by X-ray crystallography or Nuclear Magnetic Resonance. Studying 3D protein structures is a key point for better understanding protein function at a molecular level. We propose a set of accurate tools, for analysing protein structures, based on the reliable method of Voronoi-Laguerre tessellations. The Voronoi Laguerre Delaunay Protein web server (VLDPws) computes the Laguerre tessellation on a whole given system first embedded in solvent. Through this fine description, VLDPws gives the following data: (i) Amino acid volumes evaluated with high precision, as confirmed by good correlations with experimental data. (ii) A novel definition of inter-residue contacts within the given protein. (iii) A measure of the residue exposure to solvent that significantly improves the standard notion of accessibility in some cases. At present, no equivalent web server is available. VLDPws provides output in two complementary forms: direct visualization of the Laguerre tessellation, mostly its polygonal molecular surfaces; files of volumes; and areas, contacts and similar data for each residue and each atom. These files are available for download for further analysis. VLDPws can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/vldp

Aquaporins in cereals-important players in maintaining cell homeostasis under abiotic stres

Cereal productivity is reduced by environmental stresses such as drought, heat, elevated CO2, salinity, metal toxicity and cold. Sometimes, plants are exposed to multiple stresses simultaneously. Plants must be able to make a rapid and adequate response to these environmental stimuli in order to restore their growing ability. The latest research has shown that aquaporins are important players in maintaining cell homeostasis under abiotic stress. Aquaporins are membrane intrinsic proteins (MIP) that form pores in the cellular membranes, which facilitate the movement of water and many other molecules such as ammonia, urea, CO2, micronutrients (silicon and boron), glycerol and reactive oxygen species (hydrogen peroxide) across the cell and intercellular compartments. The present review primarily focuses on the diversity of aquaporins in cereal species, their cellular and subcellular localisation, their expression and their functioning under abiotic stresses. Lastly, this review discusses the potential use of mutants and plants that overexpress the aquaporin-encoding genes to improve their tolerance to abiotic stress

Determination of Ligand Pathways in Globins: Apolar Tunnels Versus Polar Gates

Background: O2 pathways in animal hemoglobins and myoglobins are controversial. Results: Ligands enter and exit sperm whale Mb and Cerebratulus lacteus Hb by completely different pathways. Conclusion: Rational mutagenesis mapping can identify ligand migration pathways and provides experimental benchmarks for testing molecular dynamics simulations. Significance: Globins can use either a polar gate or an apolar tunnel for ligand entry

The Eighth Central European Conference "Chemistry towards Biology": snapshot

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August – 1 September 2016The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August-1 September 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics

peer reviewedThe central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for E.coli in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on E. coli and S. cerevisiae comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle

INVESTIGATION OF HEME-PROTEIN INTERACTIONS IN TRUNCATED HEMOGLOBINS

Fe-protoporphyrin IX, otherwise known as heme b, is found in all hemoglobins. The heme iron is coordinated by four nitrogen atoms from the tetrapyrrole ring of protoporphyrin IX. In hemoglobin the heme is bound to the protein via a proximal histidine residue, resulting in a 5-coordinate heme. Many hemoglobins also have a sixth distal ligand in the form of an exogenous ligand, such as water or oxygen, or, in some cases, a protein residue. Despite having highly conserved tertiary structure and the same cofactor, hemoglobins display a variety of functions, such as oxygen transport, oxygen sensing, and nitric oxide scavenging. Hemoglobins and heme proteins in general are capable of controlling the chemistry performed by the heme, and therefore the function of the protein, via two primary methods: posttranslational modification (PTM) and axial ligation. In two cyanobacterial truncated hemoglobins (trHbs, from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, collectively named GlbN), when the heme contains a ferrous iron and is not carrying oxygen or carbon monoxide, a covalent bond forms between the heme-2 vinyl and a nearby histidine residue. The PTM in GlbN has been extensively studied and these studies show it is not dependent on the distal endogenous ligand and the local environment around the reactive histidine. To confirm that the main determinant of PTM is the relative positions of the reactants I engineered the PTM in the heme domain of Chlamydomonas eugametos LI637 protein (CtrHb). CtrHb is structurally similar to GlbN, sharing an average ~46% identity with them. The differences between CtrHb and GlbN are distributed throughout the structures and include some the heme pocket. Of the 21 residues that are in contact with the heme, 10 are different between the two proteins. This difference provides an altered chemical environment in which to test the reaction that generates the PTM. Enhanced chemiluminescence detection of the heme in reacted CtrHb variants shows that it is possible to engineer the PTM in this protein. This suggests that the heme pocket of GlbN does not greatly contribute to the PTM reaction and that the PTM could be engineered in other proteins. The nature of the distal heme ligand is variable and can affect the rate exogenous ligand binding, the electron transfer rate, and the redox potential of heme proteins. Owing to the importance of the distal ligand, special attention was placed on identifying the distal ligand during the optical and crystallographic characterization of the eukaryotic trHb from Chlamydomonas reinhardtii, THB1, in addition to investigating the function of this protein. In this work I expanded upon our knowledge of these two heme proteins, CtrHb and THB1, in order to gain a better understanding of how proteins control heme reactivity

Understanding substrate binding and movement in proline catabolic enzymes

"July 2014."Dissertation Supervisor: Dr. John J. Tanner.Includes vita.Proline catabolism is the breakdown of proline to glutamate. This is catalyzed by two enzymes that can be either distinct, monofunctional enzymes or fused into one bifunctional enzyme. When the two enzymes are fused together they are linked by a large, internal, water filled cavity that can channel the intermediate between the two active sites. Using crystallography, kinetics, ligand soaking, and small angle X-ray scattering, this work studied substrate binding in the active sites and movement of the intermediate between active sites within the bifunctional enzyme. This work highlighted the importance of key residues involved in substrate binding, oligomerization, product release and even intermediate trafficking. Mutations of key residues highlighted gave a more in-depth look at the path the intermediate takes in the internal chamber, by blocking off different segments of the channel. Proline is soluble at high concentrations and was used to soak crystals of proline catabolic enzymes. High concentration soaks led to movements in the enzymes that were unexpected. These high concentrations were obtainable due to proline's cryoprotective capability. This was further studied on enzymes that are not involved in proline metabolism and it was found that proline can be used, generally, as a cryoprotectant for macromolecular crystallography. The diversity of oligomeric state found in this family of states is addressed here, with plenty of data establishing the rules of higher oligomeric states found in close homologous enzymes.Includes bibliographical references

Exploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro

Includes bibliographical references.Cytochrome P450s are highly polymorphic enzymes responsible for the Phase I metabolism of over 80% of pharmaceutical drugs. Polymorphic variation can result in altered drug efficacy as well as adverse drug reactions so the lack of understanding of the effects of single amino acid substitutions on cytochrome P450 drug metabolism is a major problem for drug development. In order to begin to address this problem, this thesis describes an in silico analysis of over 300 nonsynonymous single nucleotide polymorphisms found across nine of the major human drug metabolising cytochrome P450 isoforms. Information from functional studies - in which regions of the cytochrome P450 structure important for substrate recognition, substrate and product access and egress and interaction with the cytochrome P450 reductase were delineated - was combined with in silico calculations on the effect of mutations on protein stability in order to establish the likely causes of altered drug metabolism observed for cytochrome P450 variants in functional assays carried out to date. This study revealed that 75% of all cytochrome P450 mutations showing altered activity in vitro are either predicted to be damaging to protein structure or are found within regions predicted to be important for catalytic activity. Furthermore, this study showed that 70% of the mutations that showed similar activity to the wild-type enzyme in in vitro studies lie outside of functional regions important for catalytic activity and are predicted to have no effect on protein stability. Based on these results, a cytochrome P450 polymorphic variant map was created that should find utility in predicting the functional effect of uncharacterised variants on drug metabolism. To further test the accuracy of the in silico predictions, in vitro assays were performed on a panel of CYP3A4 and CYP2C9 variants heterogeneously expressed in E.coli. All mutations predicted to alter protein function by stabilising or destabilising the apo-protein structure in silico were found to significantly alter the thermostability of the holo-protein in solution. Thermostability assays also suggest that other mutations may affect stability by disrupting haem binding, changing protein conformation or altering oligomer formation. The utility of a fluorescence-based functional P450 protein microarray platform, previously developed in our laboratory, for generating kinetic data for multiple CYP450 variants in parallel was also examined. Since the microarray platform in its current stage of development was found to be unsuitable for this purpose, kinetic data for the full panel of CYP3A4 and CYP2C9 variants was generated using solution phase assays, revealing several variants with altered catalytic turnover and/or binding affinity for fluorescent substrates