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.

Multivalency effects in Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA

The galactose specific lectin LecA partly mediates the formation of antibiotic resistant biofilms by Pseudomonas aeruginosa, an opportunistic pathogen causing lethal airways infections in immunocompromised and cystic fibrosis patients, suggesting that preventing LecA binding to natural saccharides might provide new opportunities for treatment. Here 8-fold (G3) and 16-fold (G4) galactosylated analogs of GalAG2, a tetravalent G2 glycopeptide dendrimer LecA ligand and P. aeruginosa biofilm inhibitor, were obtained by convergent chloroacetyl thioether (ClAc) ligation between 4-fold or 8-fold chloroacetylated dendrimer cores and digalactosylated dendritic arms. Hemagglutination inhibition, isothermal titration calorimetry and biofilm inhibition assays showed that G3 dendrimers bind LecA slightly better than their parent G2 dendrimers and induce complete biofilm inhibition and dispersal of P. aeruginosa biofilms, while G4 dendrimers show reduced binding and no biofilm inhibition. A binding model accounting for the observed saturation of glycopeptide dendrimer galactosyl groups and LecA binding sites is proposed based on the crystal structure of a G3 dendrimer LecA complex.

Role of Neurogranin in the regulation of calcium binding to Calmodulin

Role of Neurogranin in the regulation of calcium binding to Calmodulin Anuja Chandrasekar, B.S Advisor: M. Neal Waxham, Ph.D The overall goal of my project was to gain a quantitative understanding of how the interaction between two proteins neurogranin (RC3) and calmodulin (CaM) alters a fundamental property of CaM. CaM, has been extensively studied for more than four decades due to its seminal role in almost all biological functions as a calcium signal transducer. Calcium signals in cardiac and neuronal cells are exquisitely precise and enable activation of some processes while down-regulating others. CaM, with its four calcium binding sites, serves as a central component of calcium signaling in these cells. It is aided in this role as a regulatory hub that differentially activates targets in response to a calcium flux by proteins that alter its calcium binding properties. Neurogranin, also known as RC3, is a member of a family of small neuronal IQ (SNIQ) domain proteins that was originally thought to play a ‘capacitive’ role by sequestering CaM until a calcium influx of sufficient intensity arrived. However, based on earlier work in our lab on neurogranin, we believe that this protein plays a more nuanced role in neurons than simply acting as a CaM buffer. We believe that neurogranin is one of the proteins which, by altering the kinetics of calcium binding allow CaM to decode a variety of signals with fine precision. To quantify the interaction between CaM, neurogranin and calcium, I used biophysical techniques and computational simulations. From my results, I conclude that neurogranin finely regulates the proportion of calcium-saturated CaM and thereby directs CaM’s target specificity.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose-binding domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of 1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

The N-terminal domain of Escherichia coli enzyme I of the phosphoenolpyruvate/glycose phosphotransferase system: molecular cloning and characterization.

The bacterial phosphoenolpyruvate/glycose phosphotransferase system (PTS) comprises a group of proteins that catalyze the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to sugars concomitant with their translocation. The first two steps of the phosphotransfer sequence are PEP <--> Enzyme I (EI) <--> HPr (the histidine-containing phosphocarrier protein). We have proposed that many functions of the PTS are regulated by EI, which undergoes a monomer/dimer transition. EI monomer (63.5 kDa) comprises two major domains: a flexible C-terminal domain (EI-C) and a protease-resistant, structurally stable N-terminal domain (EI-N) containing the active site His. Trypsin treatment of Salmonella typhimurium EI yielded EI-N, designated EI-N(t). Homogeneous recombinant Escherichia coli EI-N [i.e., EI-N(r)], has now been prepared in quantity, shows the expected thermodynamic unfolding properties and, similarly to EI-N(t), is phosphorylated by phospho-HPr, but not by PEP. In addition, binding of EI-N(r) to HPr was studied by isothermal titration calorimetry: K/a = 1.4 x 10(5) M(-1) and delta H = +8.8 kcal x mol(-1). Both values are comparable to those for HPr binding to intact EI. Fluorescence anisotropy [dansyl-EI-N(r)] and gel filtration of EI-N(r) show that it does not dimerize. These results emphasize the role of EI-C in dimerization and the regulation of intact EI.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.

Synergistic action of actinoporin isoforms from the same sea anemone species assembled into functionally active heteropores

Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are pore forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families which give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here, using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other’s activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores since (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help to understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity.

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are the pore-forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families that give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here,using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other’s activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores because (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help us understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Interpretation of the temperature dependence of equilibrium and rate constants

The objective of this review is to draw attention to potential pitfalls in attempts to glean mechanistic information from the magnitudes of standard enthalpies and entropies derived from the temperature dependence of equilibrium and rate constants for protein interactions. Problems arise because the minimalist model that suffices to describe the energy differences between initial and final states usually comprises a set of linked equilibria, each of which is characterized by its own energetics. For example, because the overall standard enthalpy is a composite of those individual values, a positive magnitude for AHO can still arise despite all reactions within the subset being characterized by negative enthalpy changes: designation of the reaction as being entropy driven is thus equivocal. An experimenter must always bear in mind the fact that any mechanistic interpretation of the magnitudes of thermodynamic parameters refers to the reaction model rather than the experimental system For the same reason there is little point in subjecting the temperature dependence of rate constants for protein interactions to transition-state analysis. If comparisons with reported values of standard enthalpy and entropy of activation are needed, they are readily calculated from the empirical Arrhenius parameters. Copyright (c) 2006 John Wiley & Sons, Ltd.

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.

Thermodynamics of ligand (substrate end product) binding to endoxylanase from Chainia sp. (NCL-82-5-1): isothermal calorimetry and fluorescence titration studies

The binding of xylo-oligosaccharides to Chainia endoxylanase resulted in a decrease in fluorescence intensity of the enzyme with the formation of 1:1 complex. Equilibrium and thermodynamic parameters of ligand binding were determined by fluorescence titrations and titration calorimetry. The affinity of xylanase for the oligosaccharides increases in the order X-2 < X-3 < X-4 less than or equal to X-5. Contributions from the enthalpy towards the free energy change decreased with increasing chain length from X-2 to X-4, whereas an increase in entropy was observed, the change in enthalpy and entropy of binding being compensatory. The entropically driven binding process suggested that hydrophobic interactions as well as hydrogen bonds play a predominant role in ligand binding.