Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATR Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5 ',5 '''-P-1,P-4-tetraphosphate (AP(4)A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap(4)A bound to the active site pocket suggesting the presence of Ap(4)A synthetic activity in TdcD. Binding of Ap(4)A to the enzyme was confirmed by the structure determination of a TdcD-Ap(4)A complex obtained after cocrystallization of TdcD with commercially available Ap(4)A. Mass spectroscopic studies provided further evidence for the formation of Ap(4)A by propionate kinase in the presence of ATP. In the TdcD-Ap(4)A complex structure, Ap(4)A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.

A Study of copper-compounds by x-Ray Absorption-spectroscopy

Chemical shifts, ΔE, of the K-absorption discontinuity in several compounds of copper possessing formal oxidation states between 0 and III have been measured. The shifts show a parabolic dependence on the formal oxidation state as well as on the effective atomic charge, q, on copper. Anomalous chemical shifts shown by some of the compounds are discussed in terms of the bonding in these compounds. The ΔE values have also been correlated with the core electron binding energies obtained from X-ray photoelectron spectroscopy.

Computer modeling studies on the binding of 2',5'-linked dinucleoside phosphates to ribonuclease T1-influence of subsite interactions on the substrate specificity

The modes of binding of Gp(2',5')A, Gp(2',5')C, Gp(2',5')G and Gp(2',5')U to RNase T1 have been determined by computer modelling studies. All these dinucleoside phosphates assume extended conformations in the active site leading to better interactions with the enzyme. The 5'-terminal guanine of all these ligands is placed in the primary base binding site of the enzyme in an orientation similar to that of 2'-GMP in the RNase T1-2'-GMP complex. The 2'-terminal purines are placed close to the hydrophobic pocket formed by the residues Gly71, Ser72, Pro73 and Gly74 which occur in a loop region. However, the orientation of the 2'-terminal pyrimidines is different from that of 2'-terminal purines. This perhaps explains the higher binding affinity of the 2',5'-linked guanine dinucleoside phosphates with 2'-terminal purines than those with 2'-terminal pyrimidines. A comparison of the binding of the guanine dinucleoside phosphates with 2',5'- and 3',5'-linkages suggests significant differences in the ribose pucker and hydrogen bonding interactions between the catalytic residues and the bound nucleoside phosphate implying that 2',5'-linked dinucleoside phosphates may not be the ideal ligands to probe the role of the catalytic amino acid residues. A change in the amino acid sequence in the surface loop region formed by the residues Gly71 to Gly74 drastically affects the conformation of the base binding subsite, and this may account for the inactivity of the enzyme with altered sequence i.e., with Pro, Gly and Ser at positions 71 to 73 respectively. These results thus suggest that in addition to recognition and catalytic sites, interactions at the loop regions which constitute the subsite for base binding are also crucial in determining the substrate specificity.

Solvation and axial ligation properties of (2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetraphenylporphyrinato)zinc(II)

he solvation of (2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetraphenylporphyrinato)zinc(II)[Zn(obtpp)], in twelve different solvents results in large red shifts of the B and Q bands of the porphyrin accompanied by enhanced absorbance ratios of the Q bands. These observations are ascribed to the destabilisation of the highest occupied molecular orbital a2u of the porphyrin arising from a flow of charge from the axial ligand to the porphyrin ring through the zinc(II) ion. The binding constants of adducts of [Zn(obtpp)] with neutral bases have been found to be an order of magnitude greater than those observed for the corresponding adducts of (5,10,15,20-tetraphenylporphyrinato)-zinc and vary in the order piperidine > imidazole > pyridine > 3-methylpyridine > pyridine-3-carbaldehyde. The enhanced binding constants and large spectral shifts are interpreted in terms of the electrophilicity of [Zn(obtpp)] induced by the electron-withdrawing bromine substituents in the porphyrin core. The structure of [Zn(obtpp)(PrCN)2] has been determined; it reveals six-co-ordinated zinc(II) with two long Zn–N distance [2.51(4), 2.59(3)Å]. The porphyrin is non-planar and displays a saddle-shaped conformation.

DNA Compaction by a Dendrimer

At physiological pH, a PAMAM dendrimer is positively charged and can effectively bind negatively charged DNA. Currently, there has been great interest in understanding this complexation reaction both for fundamental (as a model for complex biological reactions) as well as for practical (as a gene delivery material and probe for sensing DNA sequence) reasons. Here, we have studied the complexation between double-stranded DNA (dsDNA) and various generations of PAMAM dendrimers (G3-05) through atomistic molecular dynamics simulations in the presence of water and ions. We report the compaction of DNA on a nanosecond time scale. This is remarkable, given the fact that such a short DNA duplex with a length close to 13 nm is otherwise thought to be a rigid rod. Using several nanoseconds long MD simulations, we have observed various binding modes of dsDNA and dendrimers for various generations of PAMAM dendrimers at varying charge ratios, and it confirms some of the binding modes proposed earlier. The binding is driven by the electrostatic interaction, and the larger the dendrimer charge, the stronger the binding affinity. As DNA wraps/binds to the dendrimer, counterions originally condensed onto DNA (Na+) and the dendrimer (Cl-) get released. We calculate the entropy of counterions and show that there is gain in entropy due to counterion release during the complexation. MD simulations demonstrate that, when the charge ratio is greater than 1 (as in the case of the G5 dendrimer), the optimal wrapping of DNA is observed. Calculated binding energies of the complexation follow the trend G5 > 04 > 03, in accordance with the experimental data. For a lower-generation dendrimer, such as G3, and, to some extent, for G4 also, we see considerable deformation in the dendrimer structure due to their flexible nature. We have also calculated the various helicoidal parameters of DNA to study the effect of dendrimer binding on the structure of DNA. The B form of the DNA is well preserved in the complex, as is evident from various helical parameters, justifying the use of the PAMAM dendrimer as a suitable delivery vehicle.

The variants of the protein toxins abrin and ricin A useful guide to understanding the processing events in the toxin transport

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1-versus-concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin-I (4.13 × 105 M−1 s−1) association with putative receptor (4-methylumbelliferyl-α-D-galactoside) is nearly two times more often than abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 1 I-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.

Dependence of the strength of interaction of carbon monoxide with transition metal clusters on the cluster size

The interaction of CO with Cu, Pd, and Ni at different coverages of the metals on solid substrates has been investigated by He II and core-level spectroscopies, after the nature of variation of the metal core-level binding energies with the coverage or the cluster size is established. The separation between the (1 pi + 5 sigma) and 4 sigma levels of CO increases with a decrease in the size of the metal clusters, accompanied by an increase in the desorption temperature. In the case of Cu, the intramolecular shakeup satellite of CO disappears on small clusters. More importantly, CO dissociates on small Ni clusters, clearly confirming that metal-CO interaction strength increases with a decrease in the cluster size.

Interaction of carbon monoxide with bimetallic overlayers

Interaction of carbon monoxide with a few chosen bimetallic overlayers has been investigated along with the core-level binding energies of the deposited metals by employing X-rays as well as UV photoelectron spectroscopies. Core-level binding energies of the deposited metals around monolayer coverages (0 similar to 1) are significantly different than those at high coverages or of the pure metals. Bimetallic overlayers such as Ni/Au and Cu/Pt showing large negative shifts in the surface core-level binding energy of the deposited metal interact strongly with carbon monoxide. In the case of Ni/Au (0(Ni) similar to 0.85), CO dissociates around 280 K. In contrast to this behavior, the interaction of CO with Pd/Mo or W, showing large positive shifts in the surface core-level binding energy, is very weak, and the CO desorption temperature is much lower than that from the clean Pd metal surface. The CO desorption temperature generally increases as the surface core-level shift of the deposited metal becomes more negative; the separation between the (5 sigma + 1 pi) and 4 sigma levels of CO also increases in this direction. These results suggest that the variation in the strength of interaction of CO with bimetallic overlayers is a chemical manifestation of the shift in the surface core-level binding energies of the deposited metals at monolayer coverages.

Pressure-induced band gap reduction, orientational ordering and reversible amorphization in single crystals of C70: Photoluminescence and Raman studies

Photoluminescence and Raman scattering experiments have been carried out on single crystals of C70 up to 31 GPa to investigate the effect of pressure on the optical band gap, vibrational modes and stability of the molecule. The photoluminescence band shifts to lower energies and the pressure dependence of the band maxima yields the hydrostatic deformation potential to be 2.15 eV. The slope changes in the pressure dependence of peak positions and linewidths of the Raman modes associated with the intramolecular vibrations at 1 GPa mark the known face-centred cubic-->rhombohedral orientational ordering transition. The reversible amorphization in C70 at P > 20 GPa has been compared with the irreversible amorphization in C60 at P > 22 GPa in terms of carbon-carbon distance between the neighbouring molecules at the threshold transition pressures, in conjunction with the interplay between the intermolecular and intramolecular interactions.

Modes of binding of 2'-AMP to RNase T1. A computer modeling study

The modes of binding of adenosine 2'-monophosphate (2'-AMP) to the enzyme ribonuclease (RNase) T1 were determined by computer modelling studies. The phosphate moiety of 2'-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites--(1) The primary base binding site where the guanine of 2'-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3'-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2'-AMP to RNase T1 were found to be of nearly the same energy implying that in solution 2'-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T1-2'-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T1 catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2'-AMP and 2'-GMP with RNase T1 reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme-2'-GMP complex.

Refolding of riboflavin carrier protein as probed by biochemical and immunological parameters

The unfolding of the chicken egg white riboflavin carrier protein by disulfide reduction with dithiothreitol led to aggregation with concomitant loss of ligand binding characteristics and the capacity to interact with six monoclonal antibodies directed against surface-exposed discontinuous epitopes. The reduced protein could, however, bind to a monoclonal antibody recognizing sequential epitope. Under optimal conditions of protein refolding, the vitamin carrier protein regained its folded structure with high efficiency with simultaneous complete restoration of hydrophobic flavin binding site as well as the epitopic conformations exposed at the surface in a manner comparable to its native form.

Photoemission study of pyrite-type transition-metal chalcogenides MS2-xSex (M=Fe, Co, Ni)

The electronic structures of pyrite-type transition-metal chalcogenides MS2-xSex (M = Fe, Co, Ni) has been investigated by photoemission and inverse-photoemission spectroscopy. The valence-band spectrum of ferromagnetic CoS2 does not show exchange splitting of the Co 3d peak, in disagreement with band-structure calculations. High-resolution photoemission spectra of NiS1.55Se0.45 shows spectral weight transfer from low (similar or equal to 50 meV) to high (0.2-0.5 eV) binding energies, in going from the metallic to the insulating phase.

Biological water: Femtosecond dynamics of macromolecular hydration

The unique features of a macromolecule and water as a solvent make the issue of solvation unconventional, with questions about the static versus dynamic nature of hydration and the, physics of orientational and translational diffusion at the boundary. For proteins, the hydration shell that covers the surface is critical to the stability of its structure and function. Dynamically speaking, the residence time of water at the surface is a signature of its mobility and binding. With femtosecond time resolution it is possible to unravel the shortest residence times which are key for the description of the hydration layer, static or dynamic. In this article we review these issues guided by experimental studies, from this laboratory, of polar hydration dynamics at the surfaces of two proteins (Subtilisin Carlsberg (SC) and Monellin). The natural probe tryptophan amino acid was used for the interrogation of the dynamics, and for direct comparison we also studied the behavior in bulk water - a complete hydration in 1 ps. We develop a theoretical description of solvation and relate the theory to the experimental observations. In this - theoretical approach, we consider the dynamical equilibrium in the hydration shell, defining the rate processes for breaking and making the transient hydrogen bonds, and the effective friction in the layer which is defined by the translational and orientational motions of water molecules. The relationship between the residence time of water molecules and the observed slow component in solvation dynamics is a direct one. For the two proteins studied, we observed a "bimodal decay" for the hydration correlation function, with two primary relaxation times: ultrafast, typically 1 ps or less, and longer, typically 15-40 ps, and both are related to the residence time at the protein surface, depending on the binding energies. We end by making extensions to studies of the denatured state of the protein, random coils, and the biomimetic micelles, and conclude with our thoughts on the relevance of the dynamics of native structures to their functions.

Systematic trends in structural and thermodynamic properties of ternary oxides in the systems Ln-Pd-O (Ln = lanthanide element)

Isothermal sections of the phase diagrams for the systems Ln-Pd-O (Ln = lanthanide element) at 1223 K indicate the presence of two inter-oxide compounds Ln(4)PdO(7) and Ln(2)Pd(2)O(5) for Ln = La, Pr, Nd, Sm, three compounds Ln(4)PdO(7), Ln(2)PdO(4) and Ln(2)Pd(2)O(5) for Ln = Eu, Gd and only one compound of Ln(2)Pd(2)O(5) for Ln = Tb to Ho. The lattice parameters of the compounds Ln(4)PdO(7), Ln(2)PdO(4) and Ln(2)Pd(2)O(5) show systematic nonlinear variation with atomic number. The unit cell volumes decrease with increasing atomic number. The standard Gibbs energies, enthalpies and entropies of formation of the ternary oxides from their component binary oxides (Ln(2)O(3) and PdO) have been measured recently using an advanced version of the solid-state electrochemical cell. The Gibbs energies and enthalpies of formation become less negative with increasing atomic number of Ln. For all the three compounds, the variation in Gibbs energy and enthalpy of formation with atomic number is markedly non-linear. The decrease in stability with atomic number is most pronounced for Ln(2)Pd(2)O(5), followed by Ln(4)PdO(7) and Ln(2)PdO(4). This is probably related to the repulsion between Pd2+ ions on the opposite phases Of O-8 cubes in Ln(2)Pd(2)O(5), and the presence of Ln-filled O-8 cubes that share three faces with each other in Ln4PdO7. The values for entropy of formation of all the ternary oxides from their component binary oxides are relatively small. Although the entropies of formation show some scatter, the average value for Ln = La, Pr, Nd is more negative than the average value for the other lanthanide elements. From this difference, an average value for the structure transformation entropy of Ln(2)O(3) from C-type to A-type is estimated as 0.87 J.mol(-1).K-1. The standard Gibbs energies of formation of these ternary oxides from elements at 1223 K are presented as a function of lanthanide atomic number. By invoking the Neumann-Kopp rule for heat capacity, thermodynamic properties of the inter-oxide compounds at 298.15 K are estimated. (C) 2002 Elsevier Science Ltd. All rights reserved.

Allosteric Communication in Cysteinyl tRNA Synthetase A NETWORK OF DIRECT AND INDIRECT READOUT

Protein structure networks are constructed for the identification of long-range signaling pathways in cysteinyl tRNA synthetase (CysRS). Molecular dynamics simulation trajectory of CysRS-ligand complexes were used to determine conformational ensembles in order to gain insight into the allosteric signaling paths. Communication paths between the anticodon binding region and the aminoacylation region have been identified. Extensive interaction between the helix bundle domain and the anticodon binding domain, resulting in structural rigidity in the presence of tRNA, has been detected. Based on the predicted model, six residues along the communication paths have been examined by mutations (single and double) and shown to mediate a coordinated coupling between anticodon recognition and activation of amino acid at the active site. This study on CysRS clearly shows that specific key residues, which are involved in communication between distal sites in allosteric proteins but may be elusive in direct structure analysis, can be identified from dynamics of protein structure networks.