Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxy) ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43*) side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl-6 and Br-8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90) by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91)) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Investigation of the interaction of alkali ions with surfactant head groups for the formation of lyotropic biaxial nematic phase via optical birefringence measurements.

Lyotropic liquid crystals exhibiting nematic phases were obtained from the mixtures potassium laurate/alkali sulfate salts (M2SO4)/1-undecanol (UndeOH)/water and sodium dodecyl sulfate (SDS)/M2SO4/1-dodecanol (DDeOH)/water, where M2SO4 represents the alkali sulfate salts being Li2SO4, Na2SO4, K2SO4, Rb2SO4 or Cs2SO4. The birefringences measurements were performed via laser conoscopy. Our results indicated that cosmotropic and chaotropic behaviors of both ions and head groups are very important to obtain lyotropic biaxial nematic phase. To obtain the biaxial nematic phase, surfactant head group and ion present in lyotropic mixture have relatively opposite behavior, e.g. one more cosmotropic (more chaotropic) other less cosmotropic (less chaotropic) or vice versa.

Theoretical investigation of the interaction of a polymer film with a nanoparticle

The fundamental aim in our investigation of the interaction of a polymer film with a nanoparticle is the extraction of information on the dynamics of the liquid using a single tracking particle. In this work two theoretical methods were used: one passive, where the motion of the particle measures the dynamics of the liquid, one active, where perturbations in the system are introduced through the particle. In the first part of this investigation a thin polymeric film on a substrate is studied using molecular dynamics simulations. The polymer is modeled via a 'bead spring' model. The particle is spheric and non structured and is able to interact with the monomers via a Lennard Jones potential. The system is micro-canonical and simulations were performed for average temperatures between the glass transition temperature of the film and its dewetting temperature. It is shown that the stability of the nanoparticle on the polymer film in the absence of gravity depends strongly on the form of the chosen interaction potential between nanoparticle and polymer. The relative position of the tracking particle to the liquid vapor interface of the polymer film shows the glass transition of the latter. The velocity correlation function and the mean square displacement of the particle has shown that it is caged when the temperature is close to the glass transition temperature. The analysis of the dynamics at long times shows the coupling of the nanoparticle to the center of mass of the polymer chains. The use of the Stokes-Einstein formula, which relates the diffusion coefficient to the viscosity, permits to use the nanoparticle as a probe for the determination of the bulk viscosity of the melt, the so called 'microrheology'. It is shown that for low frequencies the result obtained using microrheology coincides with the results of the Rouse model applied to the polymer dynamics. In the second part of this investigation the equations of Linear Hydrodynamics are solved for a nanoparticle oscillating above the film. It is shown that compressible liquids have mechanical response to external perturbations induced with the nanoparticle. These solutions show strong velocity and pressure profiles of the liquid near the interface, as well as a mechanical response of the liquid-vapor interface. The results obtained with this calculations can be employed for the interpretation of experimental results of non contact AFM microscopy

When that tune runs through your head: A PET investigation of auditory imagery for familiar melodies

The present study used positron emission tomography (PET) to examine the cerebral activity pattern associated with auditory imagery forfamiliar tunes. Subjects either imagined the continuation of nonverbaltunes cued by their first few notes, listened to a short sequence of notesas a control task, or listened and then reimagined that short sequence. Subtraction of the activation in the control task from that in the real-tune imagery task revealed primarily right-sided activation in frontal and superior temporal regions, plus supplementary motor area(SMA). Isolating retrieval of the real tunes by subtracting activation in the reimagine task from that in the real-tune imagery task revealedactivation primarily in right frontal areas and right superior temporal gyrus. Subtraction of activation in the control condition from that in the reimagine condition, intended to capture imagery of unfamiliarsequences, revealed activation in SMA, plus some left frontal regions. We conclude that areas of right auditory association cortex, together with right and left frontal cortices, are implicated in imagery for familiartunes, in accord with previous behavioral, lesion and PET data. Retrieval from musical semantic memory is mediated by structures in the right frontal lobe, in contrast to results from previous studies implicating left frontal areas for all semantic retrieval. The SMA seems to be involved specifically in image generation, implicating a motor code in this process.

When that tune runs through your head: a PET investigation of auditory imagery for familiar melodies

The present study used positron emission tomography (PET) to examine the cerebral activity pattern associated with auditory imagery for familiar tunes. Subjects either imagined the continuation of nonverbal tunes cued by their first few notes, listened to a short sequence of notes as a control task, or listened and then reimagined that short sequence. Subtraction of the activation in the control task from that in the real-tune imagery task revealed primarily right-sided activation in frontal and superior temporal regions, plus supplementary motor area (SMA). Isolating retrieval of the real tunes by subtracting activation in the reimagine task from that in the real-tune imagery task revealed activation primarily in right frontal areas and right superior temporal gyrus. Subtraction of activation in the control condition from that in the reimagine condition, intended to capture imagery of unfamiliar sequences, revealed activation in SMA, plus some left frontal regions. We conclude that areas of right auditory association cortex, together with right and left frontal cortices, are implicated in imagery for familiar tunes, in accord with previous behavioral, lesion and PET data. Retrieval from musical semantic memory is mediated by structures in the right frontal lobe, in contrast to results from previous studies implicating left frontal areas for all semantic retrieval. The SMA seems to be involved specifically in image generation, implicating a motor code in this process.

Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch-specific thymine DNA glycosylase (TDG or G/T glycosylase), which catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base per se. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2′-hydrogen atoms with fluorine in the substrate 2′-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase–DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an ≈20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3′ side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.

Detection of a Yb3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Near infrared Yb3+ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb3+ site is observed. The Yb3+ binding site in bO is observed to consist of PO2− groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb3+ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca2+ (or Mg2+) and lanthanides in phospholipid membrane proteins.

Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation.

Electron microscopic visualization indicates that the transcription activator NRI (NTRC) binds with exceptional selectivity and efficiency to a sequence-induced superhelical (spiral) segment inserted upstream of the glnA promoter, accounting for its observed ability to substitute for the natural glnA enhancer. The cooperative binding of NRI to the spiral insert leads to protein oligomerization which, at higher concentration, promotes selective coating of the entire superhelical segment with protein. Localization of NRI at apical loops is observed with negatively supercoiled plasmid DNA. With a linear plasmid, bending of DNA is observed. We confirm that NRI is a DNA-bending protein, consistent with its high affinity for spiral DNA. These results prove that spiral DNA without any homology to the NRI-binding sequence site can substitute for the glnA enhancer by promoting cooperative activator binding to DNA and facilitating protein oligomerization. Similar mechanisms might apply to other prokaryotic and eukaryotic activator proteins that share the ability to bend DNA and act efficiently as multimers.

A vindication of the authenticity of the narratives contained in the first two chapters of the Gospels of St. Matthew & St. Luke : being an investigation of objections urged by the Unitarian editors of the improved version of the New Testament ; with an appendix containing strictures on the variations between the first and fourth editions of that work /

Contains bibliographical footnotes.

Investigation of lobbying on utility holding company bills : hearings, Seventy-fourth Congress, first session, on H. Res. 288, a resolution authorizing and directing the Committee on Rules to investigate lobbying activities with respect to utility holding companies.

Hearings held July 9-17, 1935.

Investigation of illegal or improper activities in connection with the 1996 federal election campaign : hearings before the Committee on Governmental Affairs, United States Senate, One Hundred Fifth Congress, first session.

"October 22, 23, 29, and 30, 1997"--Pt. 10.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Further-more, a mode of binding interaction for the inhibitor, a-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Density functional theory investigation of 3d, 4d, and 5d 13-atom metal clusters

The knowledge of the atomic structure of clusters composed by few atoms is a basic prerequisite to obtain insights into the mechanisms that determine their chemical and physical properties as a function of diameter, shape, surface termination, as well as to understand the mechanism of bulk formation. Due to the wide use of metal systems in our modern life, the accurate determination of the properties of 3d, 4d, and 5d metal clusters poses a huge problem for nanoscience. In this work, we report a density functional theory study of the atomic structure, binding energies, effective coordination numbers, average bond lengths, and magnetic properties of the 3d, 4d, and 5d metal (30 elements) clusters containing 13 atoms, M(13). First, a set of lowest-energy local minimum structures (as supported by vibrational analysis) were obtained by combining high-temperature first- principles molecular-dynamics simulation, structure crossover, and the selection of five well-known M(13) structures. Several new lower energy configurations were identified, e. g., Pd(13), W(13), Pt(13), etc., and previous known structures were confirmed by our calculations. Furthermore, the following trends were identified: (i) compact icosahedral-like forms at the beginning of each metal series, more opened structures such as hexagonal bilayerlike and double simple-cubic layers at the middle of each metal series, and structures with an increasing effective coordination number occur for large d states occupation. (ii) For Au(13), we found that spin-orbit coupling favors the three-dimensional (3D) structures, i.e., a 3D structure is about 0.10 eV lower in energy than the lowest energy known two-dimensional configuration. (iii) The magnetic exchange interactions play an important role for particular systems such as Fe, Cr, and Mn. (iv) The analysis of the binding energy and average bond lengths show a paraboliclike shape as a function of the occupation of the d states and hence, most of the properties can be explained by the chemistry picture of occupation of the bonding and antibonding states.

Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional Biochemistry