1H n.m.r. parameters of the N-terminal 19-residue S-peptide of ribonuclease in aqueous solution

The 1H n.m.r. chemical shifts and the spin-spin coupling constants of the N-terminal 19-residue S-peptide of ribonuclease A have been measured in a 10 mM solution in D2O, pD 3.0, 27 degrees, at 300 MHz. The titration parameters for end groups Lys-1 and Ala-19 and side chains Lys-1, Glu-2, Lys-7, Glu-9, Arg-10, His-12 and Asp-14 have been determined at 90 MHz. An assignment of observed signals to individual residue protons based upon characteristic shifts, spectral analysis, double resonance, titration shifts and comparison with the spectrum of C-peptide (N-terminal 13-residue) is proposed. Differences in the observed chemical shifts, pKa's and titration shifts with reference to those proposed as "random coil" parameters are not large enough to assume the existence of a significant population of secondary structure in the conditions studied. The H alpha chemical shifts differences can be accounted for by the Phe-8 phenyl ring current for an extended peptide backbone conformation and appropriate values for the torsion angles chi 1 Phe-8 and chi 2 Phe-8.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

Self-association of adenosine 5'-monophosphate (5'-AMP) as a function of pH and in comparison with adenosine, 2'-AMP and 3'-AMP

The concentration dependence of the chemical shifts for protons H-2, H-8, and H-1' of adenosine (Ado), 2'-AMP, 3'-AMP and 5'-AMP was measured in D2O at 27 degrees C under several degrees of protonation. All results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for Ado decrease with increasing protonation: Ado (K = 15 M-1) greater than D(Ado)+/Ado (6.0 M-1) greater than D(Ado)+ (0.9 M-1). In contrast, a maximum is observed with 5'-AMP: 5'-AMP2- (K = 2.1 M-1) less than D(5'-AMP)- (3.4 M-1) less than D2(5'-AMP) +/- /D(5'-AMP)- (5.6 M-1) greater than D2(5'-AMP) +/- (approximately 2 M-1) greater than D3(5'-AMP)+ (less than or equal to 1 M-1). Self-stacking is most pronounced here if 50% of the adenine residues are protonated at N-1; complete base protonation reduces the stacking tendency drastically. Comparing the self-association of 2'-, 3'- and 5'-AMP shows that there is no influence of the phosphate-group position in the 2-fold negatively charged species, i.e., K congruent to 2 M-1 for all three AMP2- species. More importantly, there is also no significant influence observed if the stacking tendency of the three D2(AMP) +/- /D(AMP)-1:1 mixtures is compared (K congruent to 6-7 M-1); moreover, the measured association constants are within experimental error identical with the constant determined for D(Ado)+/Ado (K = 6.0 M-1). This indicates that any coulombic contribution between the -PO3(H)- group and the H+ (N-1) unit of the adenine residue to the stability of the mentioned stacks in D2O is small. However, experiments in 50% (v/v) dioxane-D8/D2O with the D2(5'-AMP) +/- /D(5'-AMP)- 1:1 system reveal, despite its low solubility, that coulombic interactions contribute to the self-association in an environment with a reduced polarity (compared to that of water). The implications of these observations for biological systems are briefly indicated.     

Secondary structural analysis of the Na(+),K(+)-ATPase and its Na(+) (E(1)) and K(+) (E(2)) complexes by FTIR spectroscopy

The Na(+),K(+)-ATPase is an integral membrane protein which transports sodium and potassium cations against an electrochemical gradient. The transport of Na(+) and K(+) ions is presumably connected to an oscillation of the enzyme between the two conformational states, the E(1) (Na(+)) and the E(2) (K(+)) conformations. The E(1) and E(2) states have different affinities for ligand interaction. However, the determination of the secondary structure of this enzyme in its sodium and potassium forms has been the subject of much controversy. This study was designed to provide a quantitative analysis of the secondary structure of the Na(+),K(+)-ATPase in its sodium (E(1)) and potassium (E(2)) states in both H(2)O and D(2)O solutions at physiological pH, using Fourier transform infrared (FTIR) with its self-deconvolution and second derivative resolution enhancement methods, as well as curve-fitting procedures. Spectroscopic analysis showed that the secondary structure of the sodium salt of the Na(+),K(+)-ATPase in H(2)O solution contains alpha-helix 19.8+/-1%, beta-sheet 25.6+/-1%, turn 9.1+/-1%, and beta-anti 7.5+/-1%, whereas in D(2)O solution, the enzyme shows alpha-helix 16.8+/-1%, beta-sheet 24.5+/-1.5%, turn 10.9+/-1%, beta-anti 9.8+/-1%, and random coil 38.0+/-2%. Similarly, the potassium salt in H(2)O solution contains alpha-helix 16.6+/-1%, beta-sheet 26.4+/-1.5%, turn 8.9+/-1%, and beta-anti 8.1+/-1%, while in D(2)O solution it shows alpha-helix 16.2+/-1%, beta-sheet 24.5+/-1.5%, turn 10.3+/-1%, beta-anti 9.0+/-1%, and random coil 40+/-2%. Thus the main differences for the sodium and potassium forms of the Na(+),K(+)-ATPase are alpha-helix 3.2% in H(2)O and 0.6% in D(2)O, beta-sheet (pleated and anti) 1.5% in H(2)O and random structure 2% (D(2)O), while for other minor components (turn structure), the differences are less than 1%.     

Effects of extrinsic imidazole ligation on the molecular and electronic structure of cytochrome c

Although imidazole ligand binding to cytochrome c is not directly related to its physiological function, it has the potential to provide valuable information on the molecular and electronic structure of the protein. The solution structure of the imidazole adduct of oxidized horse heart cytochrome c (Im-cyt c) has been determined through 2D NMR spectroscopy. The Im-cyt c, 8 mM in 1.2 M imidazole solution at pH 5.7 and 313 K, provided altogether 2,542 NOEs (1,901 meaningful NOEs) and 194 pseudocontact shifts. The 35 conformers of the family show the RMSD values to the average structure of 0.063+/-0.007 nm for the backbone and 0.107+/-0.007 nm for all heavy atoms, respectively. The characterization of Im-cyt c is discussed in detail both in terms of structure and electronic properties. The replacement of the axial ligand Met80 with the exogenous imidazole ligand induces significant conformation changes in both backbone and side chains of the residues located in the distal axial ligand regions. The imidazole ligand binds essentially parallel to the imidazole of the proximal histidine, the two planes forming an angle of 8+/-7 degrees. The electron delocalization on the heme moiety and the magnetic susceptibility tensor are consistent with these structural features.     

Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH-dependent current fluctuations.

To probe protonation dynamics inside the fully open alpha-toxin ion channel, we measured the pH-dependent fluctuations in its current. In the presence of 1 M NaCl dissolved in H2O and positive applied potentials (from the side of protein addition), the low frequency noise exhibited a single well defined peak between pH 4.5 and 7.5. A simple model in which the current is assumed to change by equal amounts upon the reversible protonation of each of N identical ionizable residues inside the channel describes the data well. These results, and the frequency dependence of the spectral density at higher frequencies, allow us to evaluate the effective pK = 5.5, as well as the rate constants for the reversible protonation reactions: kon = 8 x 10(9) M-1 s-1 and koff = 2.5 x 10(4) s-1. The estimate of kon is only slightly less than the diffusion-limited values measured by others for protonation reactions for free carboxyl or imidazole residues. Substitution of H2O by D2O caused a 3.8-fold decrease in the dissociation rate constant and shifted the pK to 6.0. The decrease in the ionization rate constants caused by H2O/D2O substitution permitted the reliable measurement of the characteristic relaxation time over a wide range of D+ concentrations and voltages. The dependence of the relaxation time on D+ concentration strongly supports the first order reaction model. The voltage dependence of the low frequency spectral density suggests that the protonation dynamics are virtually insensitive to the applied potential while the rate-limiting barriers for NaCl transport are voltage dependent. The number of ionizable residues deduced from experiments in H2O (N = 4.2) and D2O (N = 4.1) is in good agreement.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

pH-dependence of 13C chemical shifts and 13C,H coupling constants in imidazole and L-histidine.

The pH-dependence of selected 13C chemical shifts reflects the state of ionization of the imidazole ring in both imidazole and L-histidine. Titration of the amino and carboxyl groups of histidine also perturbs the shifts. The coupling constants 1J (13C(2),H) and 1J (13C(5),H) for both compounds also vary with pH, but in L-histidine these constants are relatively insensitive to the titration of groups outside the imidazole ring.     

Solution structure and dynamic character of the histidine-containing phosphotransfer domain of anaerobic sensor kinase ArcB from Escherichia coli

An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions. Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPt(ArcB)), which has a phosphorylation site, His717. The backbone dynamics were also investigated by analyses of the (15)N relaxation data and amide hydrogen exchange rates. Furthermore, the protonation states of the histidine imidazole rings were characterized by means of (1)H and (15)N chemical shifts at various pHs. The determined solution structure of HPt(ArcB) contains five helices and forms a four-helix bundle motif like other HPt domains. The obtained order parameters, S (2), [(1)H]-(15)N heteronuclear NOE values, and chemical exchange parameters, R(ex), showed that the alpha-helical regions of HPt(ArcB) are rigid on both picosecond to nanosecond and microsecond to millisecond time scales. On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D. These results suggest that HPt(ArcB) may undergo a small conformational change in helix D upon phosphorylation. It was also shown that the imidazole ring of His717 has a pK(a) value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other. This is consistent with the solution structure of HPt(ArcB), in which the imidazole ring of His717 is exposed to the solvent.     

15N- and 1H-NMR investigations of the active-site amino acids in semisynthetic RNase S' and RNase A

Extensive 15N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively 15N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The 15N resonances of the side chains of lysine-7 (N epsilon), glutamine-11 (N gamma), and histidine-12 (N pi, tau) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and 5'AMP. The analysis of the 15N-1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the N tau signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange. For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 1H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8-6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.     

Carbon-13 magnetic resonance spectra of nucleosides and their Pd(II) complexes.

Chemical shifts occurring in carbon-13 magnetic resonance spectroscopy are utilized to assess the site of complexation of nucleosides to enPdC12 in neutral aqueous solutions. Binding occurs at N3 in cytidine, thymidine, and uridien, at N7 in 1-methylguanosine, and at N1 in guanosine. For most carbon atoms adjacent to N3 in the pyrimidine nucleosides the complexation shifts of the basic ligand are about 30% of the corresponding upfield protonation shifts. All complexes are of the form enPdL2 indicating that the ligands are unidentate and that the tendency to chelation is weak. Carbon-13 magnetic resonance spectroscopy appears to be the best method for delineating these complexes in solution. Due to the high avidity of chloride ion for Pt(II), cis dichloro Pd(II) complexes may be better models for intracellular action of the corresponding Pt(II) complexes than the Pt(II) complexes themselves.     

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.     

Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.     

NMR determination of the conformational and drug binding properties of the DNA heptamer d(GpCpGpApApGpC) in aqueous solution.

1D and 2D NMR spectroscopy (500/600 MHz) has been used to investigate the equilibrium conformational states of the deoxyheptanucleotide 5'-d(GpCpGpApApGpC), as well as its complexation with the phenanthridinium drug ethidium bromide (EB). Quantitative determination (reaction constants and thermodynamic parameters) of the conformational equilibrium of the heptamer in solution and its complexation with EB was based on analysis of the dependence of proton chemical shifts on concentration (at two temperatures, 298 and 308 K) and on temperature (in the range 278-353 K). The experimental results were analysed in terms of a model of the dynamic equilibrium between single-stranded, hairpin and bulged dimer forms of the deoxyheptanucleotide and its complexes with EB. Calculation of the relative amounts of the different complexes reveals important features of the dynamic equilibrium as a function of both temperature and the ratio of the drug and heptamer concentrations. The quantitative analysis also provides the limiting proton chemical shifts of EB in each complex which have been used to determine the most favourable structures of the intercalated complexes of EB with the (GC) sites of both the hairpin and dimer forms of the heptanucleotide.     

NMR investigation of the complexation of daunomycin with deoxytetranucleotides of different base sequence in aqueous solution

500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5'-d(CGCG), 5'-d(GCGC), 5'-d(TGCA), 5'-d(ACGT) and 5'-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H - 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies deltaH, entropies deltaS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the conformational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution.     

Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes

The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.     

Coordination chemistry of co(II)-bleomycin: its investigation through NMR and molecular dynamics

Previous studies on the coordination chemistry of Co-bleomycin have suggested the secondary amine in beta-aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole rings, respectively, and the amide nitrogen in beta-hydroxyhistidine as equatorial ligands to the cobalt ion. The primary amine in beta-aminoalanine and the carbamoyl group of the mannose have been proposed alternatively as possible axial ligands. The first coordination sphere of Co(II) in Co(II)BLM has been investigated in the present study through the use of NMR and molecular dynamics calculations. The data collected from the NMR experiments are in agreement with the equatorial ligands previously proposed, and also support the participation of the primary amine as an axial ligand. The paramagnetic shifts of the gulose and mannose protons could suggest the latter as a second axial ligand. This possibility was investigated by way of molecular dynamics, with distance restraints derived from the relaxation times measured through NMR. The molecular dynamics results indicate that the most favorable structure is six-coordinate, with the primary amine and either the carbamoyl oxygen or a solvent molecule occupying the axial sites. The analysis of the structures previously derived for HOO-Co(III)-bleomycin and HOO-Co(III)-pepleomycin led us to propose the six-coordinate structure with only endogenous ligands, as the one held in solution by the Co(II) derivative of bleomycin.     

Specific interactions of mercury chloride with membranes and other ligands as revealed by mercury-NMR.

High resolution mercury nuclear magnetic resonance (199Hg-NMR) experiments have been performed in order to monitor mercury chemical speciation when HgCl2 is added to water solutions and follow mercury binding properties towards biomembranes or other ligands. Variations of 199Hg chemical shifts by several hundred ppm depending upon pH and/or pCl changes or upon ligand or membrane addition afforded to determine the thermodynamic parameters which describe the equilibria between the various species in solution. By comparison to an external reference, the decrease in concentration of mercury species in solution allowed to estimate the amount as well as the thermodynamic parameters of unlabile mercury-ligand or mercury-membrane complexes. Hence, some buffer molecules can be classified in a scale of increasing complexing power towards Hg(II): EGTA greater than Tris greater than HEPES. In contrast, MOPS, Borax, phosphates and acetates show little complexation properties for mercury, in our experimental conditions. Evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes has been found. Hg(II) does not form complexes with egg phosphatidylcholine membranes. Interaction with PE and PS model membranes can be described by the presence of two mercury sites, one labile, the other unlabile, in the NMR time scale. In the labile site Hg(PE) and Hg(PS)2 would be formed whereas in the unlabile site Hg(II) would establish bridges between three PE or PS molecules. Calculated thermodynamic data clearly indicate that PE is a better complexing agent than PS. Evidence is also found that complexation with lipids uses at first the HgCl2 species. Interestingly, mercury complexation with ligands or membranes can be completely reversed by addition of decimolar NaCl solutions. Minute mechanisms for mercury complexation with the primary amine of PE or PS membrane head groups are discussed.     

Design of aminated poly(1-vinylimidazole) for a new pH-sensitive polycation to enhance cell-specific gene delivery

Poly(1-vinylimidazole) (PVIm) with aminoethyl groups has been synthesized as a new pH-sensitive polycation to enhance cell-specific gene delivery. The resulting aminated PVIm (PVIm-NH2) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurement. Hemolysis assay showed that PVIm-NH2 enhanced membrane disruptive ability at endosomal pH, owing to the protonation of the imidazole groups with a pKa value around 6.0. Agarose gel retardation assay proved that the introduced aminoethyl groups worked as anchor groups to retain DNA. Furthermore, the ternary complex of DNA, PVIm-NH2, and a poly(L-lysine) conjugated with lactose molecules, PLL-Lac, at pH 7.4 dissociated the PLL-Lac polycation by protonation of the imidazole groups of PVIm-NH2 at pH 6.0. The resulting PVIm-NH2/DNA binary complexes easily released DNA, as compared with the PLL-Lac/PVIm-NH2/DNA ternary complex, which was examined by competitive exchange with dextran sulfate. By using PVIm-NH2 as a pH-sensitive DNA carrier, as well as PLL-Lac as a cell-targeting DNA carrier, the resulting ternary complex specifically mediated the gene expression, which depended on the protonation of the imidazole groups, on human hepatoma HepG2 cells with asialoglycoprotein receptors. These results suggest that the cell-specific gene delivery mediated by PLL-Lac was enhanced by PVIm-NH2 as a new pH-sensitive polycation.