Flexibility of a biotinylated ligand in artificial metalloenzymes based on streptavidin—an insight from molecular dynamics simulations with classical and ab initio force fields

In the field of enzymatic catalysis, creating activity from a non catalytic scaffold is a daunting task. Introduction of a catalytically active moiety within a protein scaffold offers an attractive means for the creation of artificial metalloenzymes. With this goal in mind, introduction of a biotinylated d6-piano-stool complex within streptavidin (SAV) affords enantioselective artificial transfer-hydrogenases for the reduction of prochiral ketones. Based on an X-ray crystal structure of a highly selective hybrid catalyst, displaying significant disorder around the biotinylated catalyst [η6-(p-cymene)Ru(Biot-p-L)Cl], we report on molecular dynamics simulations to shed light on the protein–cofactor interactions and contacts. The results of these simulations with classical force field indicate that the SAV-biotin and SAV-catalyst complexes are more stable than ligand-free SAV. The point mutations introduced did not affect significantly the overall behavior of SAV and, unexpectedly, the P64G substitution did not provide additional flexibility to the protein scaffold. The metal-cofactor proved to be conformationally flexible, and the S112K or P64G mutants proved to enhance this effect in the most pronounced way. The network of intermolecular hydrogen bonds is efficient at stabilizing the position of biotin, but much less at fixing the conformation of an extended biotinylated ligand. This leads to a relative conformational freedom of the metal-cofactor, and a poorly localized catalytic metal moiety. MD calculations with ab initio potential function suggest that the hydrogen bonds alone are not sufficient factors for full stabilization of the biotin. The hydrophobic biotin-binding pocket (and generally protein scaffold) maintains the hydrogen bonds between biotin and protein

Computational modelling of metal-mediated protein-ligand interactions

Although metalloproteins account for nearly half of all proteins in nature, computational modelling of metal-mediated protein-ligand interactions is understudied and molecular mechanics programs and force field parameters compatible to proteins and transition metals are not readily available. Within this thesis, various computational approaches were pursued towards the design of artificial metalloenzymes and the modelling of metal- mediated protein ligand interactions. Four challenges were identified and addressed. The first consisted of the identification of suitable protein scaffolds for the creation of artificial facial-triad motifs. The second part focused on the development of reliable force field parameters for the anchoring of sulfonamide bearing anchors within human carbonic anhydrase 2. In order to reliably predict the geometry of catalytically relevant piano stool artificial cofactors within host proteins, the third part aimed at developing force-field parameters (using the Valbond formalism) for d6 -piano stool complexes. Finally, the fourth step combined the above developments towards the reliable prediction of first and second coordination sphere environments around artificial cofactors/inhibitors

Computational strategies for the design of new enzymatic functions

In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels–Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on molecular dynamics simulations with Quantum Mechanics/Molecular Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.This work was supported by the Spanish Ministerio de Economía y Competitividad for project CTQ2012-36253-C03, Universitat Jaume I – Spain (project P1·1B2014-26), Generalitat Valenciana – Spain (PROMETEOII/2014/022 and ACOMP/2014/277 projects), Polish National Center for Science (NCN) (grant 2011/02/A/ST4/00246, 2012−2017), the Polish Ministry of Science and Higher Education (“Iuventus Plus” program project no. 0478/IP3/2015/73, 2015-2016) and the USA National Institute of Health (ref. NIH R01 GM065368). Authors acknowledge computational resources from the Servei d’Informàtica of Universitat de València on the ‘Tirant’ supercomputer and the Servei d’Informat̀ica of Universitat Jaume I

Electronic Effects in Oxidation Reactions Utilizing Dinuclear Copper Complexes with the Bis[3-(2-hydroxybenzylideneamino)phenyl] Sulfone Ligand

Copper acetate and the ligands bis[3-(3-tert-butyl-2-hydroxy-5-methoxybenzylideneamino)phenyl] sulfone and bis[3-(3,5-di-tert-butyl-2-hydroxybenzylideneamino)phenyl] sulfone were reacted to form the complexes with 2:1 copper:ligand ratio, Cu2[B(t-Bu) (OMe)BAPS](µ-OCH3)2 (4) and with 2:2 copper:ligand ratio, Cu2[B(t-Bu)2BAPS]2 (5), respectively. Structures of 4 and 5 were determined based on IR, UV-Vis, and FAB-MS data in comparison with previously characterized related copper complexes. The two complexes 4 and 5 were utilized in the oxidation of the substrates 2,4- and 2,6-di-tertbutylphenol (dtbp) at -50C with H2O2 in CH2Cl2. The coupling products are preferred in both cases. For 2,4-dtbp, yields of 4,600% and 7,200% of 3,3’,5,5’-tetra-tert-butyl-2,2’- biphenol were achieved with the use of 4 and 5, respectively. For 2,6-dtbp, yields of 1,900% and 400% of 3,3’,5,5’-tetra-tert-butyl-4,4’-biphenol were realized utilizing 4 and 5, respectively. These show that the methoxy groups activated the complex. Based on low temperature UV-vis results, a µ-η2 :η2-peroxo or a µ-hydroperoxo intermediate was possibly formed by the reaction of 4 with the H2O2. This effected the oxidation of the 2,4- and 2,6- dtbp substrates but also resulted in the attack of other complexes which acted as substrates. A proposed oxidation mechanism using complex 4 and related complexes is presented

Protein Engineering of Microbial Ferritins

Nanotechnology has become a field of intense interest due to its applications in medicine, cosmetics, energy, and fabrics. The field has evolved to include nanodimensional self-assembling cage proteins which are multisubunit proteins surrounding a hollow cavity. Cage proteins can be engineered with precision, by either chemical, enzymatic or recombinant DNA methods, to accommodate non-native guest molecules both internally or externally. The inside cavity could be engineered to encapsulate inorganic nanoparticles or drugs, enzymes, or to serve as a size constraint reaction vessel for nanoparticle synthesis. However, exterior modification of these proteins has not been extensively explored. The most common initial studies have included the attachment of antibodies for targeting. We show that: (1) The N-termini of the subunits of Escherichia coli bacterioferritin (Bfr) can be modified by recombinant DNA methodologies to produce peptide fusions, which are termed “QTag-Bfr” tags, and which can be modified by bacterial transglutaminase to link substituted amines to the exterior surface of Bfr. These modifiable tags may be employed in the future for affinity chromatography applications, as precursors to nanoparticle superlattices and as anticancer drug/imaging agent delivery vehicles. The exact sequence of these tags was found to also control the final quaternary structure of Bfr. (2) The interior surface can be modified with affinity tags to allow for controllable encapsulation of functionalized guest molecules, and we extend previous research by elaborating on the incorporation of green fluorescent protein, gold nanoparticles, and micelles with Bfr and the “QTag-Bfr” constructs. (3) Similar strategies were extended to a recently discovered thermophilic Archaeoglobus fulgidus ferritin (AfFtn), and various engineered AfFtn were studied with respect to the encapsulation of a range of guest molecules, yet also provide a more open cavity capable of novel investigations on guest entrapment. (4) An extensive computational docking study was undertaken on the Bfr heme cofactor binding site which led to the identification of potential non-heme replacements which were tested for control of the quaternary structure of E. coli Bfr. These studies provide several versatile nanodimensional capsule protein frameworks which could be utilized in the future for the production of new biomaterials, as therapeutics in medicine and expand our knowledge on capsule protein biotemplating and protein engineering

Non-covalent interactions in organotin(IV) derivatives of 5,7-ditertbutyl- and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine as recognition motifs in crystalline self- assembly and their in vitro antistaphylococcal activity

Non-covalent interactions are known to play a key role in biological compounds due to their stabilization of the tertiary and quaternary structure of proteins [1]. Ligands similar to purine rings, such as triazolo pyrimidine ones, are very versatile in their interactions with metals and can act as model systems for natural bio-inorganic compounds [2]. A considerable series (twelve novel compounds are reported) of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl- 1,2,4-triazolo[1,5-a]pyrimidine (dptp) were synthesized and investigated by FT-IR and 119Sn M\uf6ssbauer in the solid state and by 1H and 13C NMR spectroscopy, in solution [3]. The X-ray crystal and molecular structures of Et2SnCl2(dbtp)2 and Ph2SnCl2(EtOH)2(dptp)2 were described, in this latter pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the -OH group of the ethanol moieties. The network of hydrogen bonding and aromatic interactions involving pyrimidine and phenyl rings in both complexes drives their self-assembly. Noncovalent interactions involving aromatic rings are key processes in both chemical and biological recognition, contributing to overall complex stability and forming recognition motifs. It is noteworthy that in Ph2SnCl2(EtOH)2(dptp)2 \u3c0\u2013\u3c0 stacking interactions between pairs of antiparallel triazolopyrimidine rings mimick basepair interactions physiologically occurring in DNA (Fig.1). M\uf6ssbauer spectra suggest for Et2SnCl2(dbtp)2 a distorted octahedral structure, with C-Sn-C bond angles lower than 180\ub0. The estimated angle for Et2SnCl2(dbtp)2 is virtually identical to that determined by X-ray diffraction. Ph2SnCl2(EtOH)2(dptp)2 is characterized by an essentially linear C-Sn-C fragment according to the X-ray all-trans structure. The compounds were screened for their in vitro antibacterial activity on a group of reference staphylococcal strains susceptible or resistant to methicillin and against two reference Gramnegative pathogens [4] . We tested the biological activity of all the specimen against a group of staphylococcal reference strains (S. aureus ATCC 25923, S. aureus ATCC 29213, methicillin resistant S. aureus 43866 and S. epidermidis RP62A) along with Gram-negative pathogens (P. aeruginosa ATCC9027 and E. coli ATCC25922). Ph2SnCl2(EtOH)2(dptp)2 showed good antibacterial activity with a MIC value of 5 \u3bcg mL-1 against S. aureus ATCC29213 and also resulted active against methicillin resistant S. epidermidis RP62A

The structural basis of RNA-catalyzed RNA polymerization

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.Cataloged from PDF version of thesis.Includes bibliographical references.The Class I ligase is an artificial ribozyme that catalyzes a reaction chemically identical to a single turnover of RNA-dependent RNA polymerization. Such an activity would have been requisite for the emergence of a self-replicase ribozyme, an enzyme that, according to the RNA World hypothesis, would be fundamental for the emergence of life. Demonstrating the plausibility of RNA-catalyzed self-replication, the Class I ligase catalytic machinery was previously harnessed to produce general RNA polymerase ribozymes. Hence, this ligase represents a robust model system for studying both the potential role RNA may have played in the origins of life and RNA catalysis in general. Through a combination of crystallographic and biochemical experiments, we have sought to elucidate the structure and mechanism of this ribozyme. As a starting point for our experiments, the crystal structure of the self-ligated product was solved to 3.0 Angstrom resolution, revealing a tripodal architecture in which three helical domains converge in the vicinity of the ligation junction. A handful of tertiary interactions decorate this tripod scaffold; among them were two instances of a novel motif, the A-minor triad. The structure elucidated interactions that recognize and bind the primer-template duplex and those that position the reaction electrophile. It furthermore revealed functional groups that compose the active site. Biochemical evidence and the position of these groups lead us to propose a reaction mechanism similar to that used by proteinaceous polymerases. Using a slowly reacting mutant, 3.05-3.15 Angstrom crystal structures were solved of unreacted, kinetically trapped ligase-substrate complexes bound to different metal ions. Comparison of the Ca2+- and Mg2+-bound structures explains the preference of the ligase for Mg 2+. Moreover, these structures revealed features missing in the product structure: interactions to the 5'-triphosphate and an active site catalytic metal ion. While this metal is positioned in a manner similar to the canonical "Metal A" of proteinaceous polymerases, the role of "Metal B" might have been supplanted by functional groups on the RNA. Kinetic isotope experiments and atomic mutagenesis of two active site functional groups imply that they may act in concert to electrostatically aid transition-state stabilization.by David M. Shechner.Ph.D

National synchrotron light source. Activity report, October 1, 1995--September 30, 1996

The hard work done by the synchrotron radiation community, in collaboration with all those using large-scale central facilities during 1995, paid off in FY 1996 through the DOE`s Presidential Scientific Facilities Initiative. In comparison with the other DOE synchrotron radiation facilities, the National Synchrotron Light Source benefited least in operating budgets because it was unable to increase running time beyond 100%-nevertheless, the number of station hours was maintained. The major thrust at Brookhaven came from a 15% increase in budget which allowed the recruitment of seven staff in the beamlines support group and permitted a step increment in the funding of the extremely long list of upgrades; both to the sources and to the beamlines. During the December 1995 shutdown, the VUV Ring quadrant around U10-U12 was totally reconstructed. New front ends, enabling apertures up to 90 mrad on U10 and U12, were installed. During the year new PRTs were in formation for the infrared beamlines, encouraged by the investment the lab was able to commit from the initiative funds and by awards from the Scientific Facilities Initiative. A new PRT, specifically for small and wide angle x-ray scattering from polymers, will start work on X27C in FY 1997 and existing PRTs on X26C and X9B working on macromolecular crystallography will be joined by new members. Plans to replace aging radio frequency cavities by an improved design, originally a painfully slow six or eight year project, were brought forward so that the first pair of cavities (half of the project for the X-Ray Ring) will now be installed in FY 1997. Current upgrades to 350 mA initially and to 438 mA later in the X-Ray Ring were set aside due to lack of funds for the necessary thermally robust beryllium windows. The Scientific Facilities Initiative allowed purchase of all 34 windows in FY 1996 so that the power upgrade will be achieved in FY 1997