Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.

Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: Implications to tetramer stability and ligand-binding

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis. (C) 2010 Elsevier Inc. All rights reserved.

Structural organization of cetyltrimethylammonium sulfate in aqueous solution: the effect of Na2SO4

We used dynamic light scattering (DLS), a steady-state fluorescence, time resolved fluorescence quenching (TRFQ), tensiometry, conductimetry, and isothermal titration calorimetry (ITC) to investigate the self-assembly of the cationic surfactant cetyltrimethylammonium sulfate (CTAS) in aqueous solution, which has SO42- as divalent counterion. We obtained the critical micelled concentration (cmc), aggregation number (N-agg), area per monomer (a(0)), hydrodynamic radius (R-H), and degree of counterion dissociation (alpha) of CTAS micelles in the absence and presence of up to 1 M Na2SO4 and at temperatures of 25 and 40 degrees C. Between 0.01 and 0.3 M salt the hydrodynamic radius of CTAS micelle R-H approximate to 16 angstrom is roughly independent on Na2SO4 concentration; below and above this concentration range R-H increases steeply with the salt concentration, indicating micelle structure transition, from spherical to rod-like structures. R-H increases only slightly as temperature increases from 25 to 40 degrees C, and the cmc decreases initially very steeply with Na2SO4 concentration up to about 10 mM, and thereafter it is constant. The area per surfactant at the water/air interface, a(0), initially increases steeply with Na2SO4 concentration, and then decrases above ca. 10 mM. Conductimetry gives alpha = 0.18 for the degree of counterion dissociation, and N-agg obtained by fluorescence methods increases with surfactant concentration but it is roughly independent of up to 80 mM salt. The ITC data yield cmc of 0.22 mM in water, and the calculated enthalpy change of micelle formation, Delta H-mic = 3.8 kJ mol(-1), Gibbs free energy of micellization of surfactant molecules, Delta G(mic) = -38.0 kJ mol(-1) and entropy T Delta S-mic = 41.7 kJ mol(-1) indicate that the formation of CTAS micelles is entropy-driven. (c) 2006 Elsevier B.V. All rights reserved.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

Interação de poli(etileno glicol) com brometo de dioctadecildimetilamônio e didodecilmetilamônio em dispersões aquosas

Pós-graduação em Biofísica Molecular - IBILCE

INVESTIGAÇÃO ELETROQUÍMICA E CALORIMÉTRICA DA INTERAÇÃO DE NOVOS AGENTES ANTITUMORAIS BISCATIÔNICOS COM DNA

ELECTROCHEMICAL AND CALORIMETRIC INVESTIGATION OF INTERACTION OF NOVEL BISCATIONIC ANTICANCER AGENTS WITH DNA. Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-alpha,omega-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Flavonoid interactions with human transthyretin: Combined structural and thermodynamic analysis

Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for ITRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities. (C) 2012 Elsevier Inc. All rights reserved.

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Pentafluorophenyl phenyl interaction in biphenyl DNA

We prepared and investigated oligonucleotide duplexes of the sequence d(GATGAC(X)(n)GCTAG)d(CTAGC(Y)(n)GTCATC), in which X and Y designate biphenyl- (bph) and pentafluorobiphenyl- ((5F)bph) C-nucleotides, respectively, and n varies from 0-4. These hydrophobic base substitutes are expected to adopt a zipperlike, interstrand stacking motif, in which not only bph/bph or (5F)bph/(5F)bph homo pairs, but also (5F)bph/bph mixed pairs can be formed. By performing UV-melting curve analysis we found that incorporation of a single (5F)bph/(5F)bph pair leads to a duplex that is essentially as stable as the unmodified duplex (n=0), and 2.4 K more stable than the duplex with the nonfluorinated bph/bph pair. The T(m) of the mixed bph/(5F)bph pair was in between the T(m) values of the respective homo pairs. Additional, unnatural aromatic pairs increased the T(m) by +3.0-4.4 K/couple, irrespective of the nature of the aromatic residue. A thermodynamic analysis using isothermal titration calorimetry (ITC) of a series of duplexes with n=3 revealed lower (less negative) duplex formation enthalpies (DeltaH) in the (5F)bph/(5F)bph case than in the bph/bph case, and confirmed the higher thermodynamic stability (DeltaG) of the fluorinated duplex, suggesting it to be of entropic origin. Our data are compatible with a model in which the stacking of (5F)bph versus bph is dominated by dehydration of the aromatic units upon duplex formation. They do not support a model in which van der Waals dispersive forces (induced dipoles) or electrostatic (quadrupole) interactions play a dominant role

Oligodeoxynucleotide Duplexes Containing (5′S)-5′-C-Alkyl-Modified 2′-Deoxynucleosides: Can an Alkyl Zipper across the DNA Minor-Groove Enhance Duplex Stability?

10.1002/hlca.200390311.abs A series of oligonucleotides containing (5′S)-5′-C-butyl- and (5′S)-5′-C-isopentyl-substituted 2′-deoxyribonucleosides were designed, prepared, and characterized with the intention to explore alkyl-zipper formation between opposing alkyl chains across the minor groove of oligonucleotide duplexes as a means to modulate DNA-duplex stability. From four possible arrangements of the alkyl groups that differ in the density of packing of the alkyl chains across the minor groove, three (duplex types I–III, Fig. 2) could experimentally be realized and their duplex-forming properties analyzed by UV-melting curves, CD spectroscopy, and isothermal titration calorimetry (ITC), as well as by molecular modeling. The results show that all arrangements of alkyl residues within the minor groove of DNA are thermally destabilizing by 1.5–3°/modification in Tm. We found that, within the proposed duplexes with more loosely packed alkyl groups (type-III duplexes), accommodation of alkyl residues without extended distorsion of the helical parameters of B-DNA is possible but does not lead to higher thermodynamic stability. The more densely packed and more unevenly distributed arrangement (type-II duplexes) seems to suffer from ecliptic positioning of opposite alkyl groups, which might account for a systematic negative contribution to stability due to steric interactions. The decreased stability in the type-III duplexes described here may be due either to missing hydrophobic interactions of the alkyl groups (not bulky enough to make close contacts), or to an overcompensation of favorable alkyl-zipper formation presumably by loss of structured H2O in the minor groove.

Specific binding of the mammalian high mobility group protein AT-hook 2 to the minor groove of AT -rich DNAs: Thermodynamic and specificity studies

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small transcriptional factor involved in cell development and oncogenesis. It contains three "AT-hook" DNA binding domains, which specifically recognize the minor groove of AT-rich DNA sequences. It also has an acidic C-terminal motif. Previous studies showed that HMGA2 mediates all its biological effects through interactions with AT-rich DNA sequences in the promoter regions. In this dissertation, I used a variety of biochemical and biophysical methods to examine the physical properties of HMGA2 and to further investigate HMGA2's interactions with AT-rich DNA sequences. The following are three avenues perused in this study: (1) due to the asymmetrical charge distribution of HMGA2, I have developed a rapid procedure to purify HMGA2 in the milligram range. Preparation of large amounts of HMGA2 makes biophysical studies possible; (2) Since HMGA2 binds to different AT-rich sequences in the promoter regions, I used a combination of isothermal titration calorimetry (ITC) and DNA UV melting experiment to characterize interactions of HMGA2 with poly(dA-dT) 2 and poly(dA)poly(dT). My results demonstrated that (i) each HMGA2 molecule binds to 15 AT bp; (ii) HMGA2 binds to both AT DNAs with very high affinity. However, the binding reaction of HMGA2 to poly(dA-dT) 2 is enthalpy-driven and the binding reaction of HMGA2 with poly(dA)poly(dT) is entropy-driven; (iii) the binding reactions are strongly depended on salt concentrations; (3) Previous studies showed that HMGA2 may have sequence specificity. In this study, I used a PCR-based SELEX procedure to examine the DNA binding specificity of HMGA2. Two consensus sequences for HMGA2 have been identified: 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These consensus sequences have a unique feature: the first five base pairs are AT-rich, the middle four to five base pairs are GC-rich, and the last five to six base pairs are AT-rich. All three segments are critical for high affinity binding. Replacing either one of the AT-rich sequences to a non-AT-rich sequence causes at least 100-fold decrease in the binding affinity. Intriguingly, if the GC-segment is substituted by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step towards finding its binding sites within the genome.

Structural Basis of RNA Recognition by Mycobacterium tuberculosis MazF Homologues

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolysaccharides and lipid A

Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities. The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar affinity. These interactions are driven primarily by a favourable change in entropy (delta S) and are endothermic in nature. These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, delta Cp, of -2385 J.mol-1.degree-1 for PmB-LPS interactions while the binding of PmB to lipid A displays a delta Cp of -2259 J.mol-1.degree-1. The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS.