Computational analysis,structural modeling and ligand binding site prediction of Plasmodium falciparum 1-deoxy-d-xylulose-5-phosphate synthase

Malaria remains one of the most serious infectious diseases in the world. There are five human species of the Plasmodium genus, of which Plasmodium falciparum is the most virulent and responsible for the vast majority of malaria related deaths. The unique biochemical processes that exist in Plasmodium falciparum provide a useful way to develop novel inhibitors. One such biochemical pathway is the methyl erythritol phosphate pathway (MEP), required to synthesize isoprenoid precursors. In the present study, a detailed computational analysis has been performed for 1-deoxy-d-xylulose-5-phosphate synthase, a key enzyme in MEP. The protein is found to be stable and residues from 825 to 971 are highly conserved across species. The homology model of the enzyme is developed using three web-based servers and Modeller software. It has twelve disordered regions indicating its druggability. Virtual screening of ZINC database identifies ten potential compounds in thiamine diphosphate binding region of the enzyme.     

Segment identification of a ligand binding with a protein receptor using multidimensional T1rho-, diffusion-filtered and diffusion-ordered NOESY experiments.

Multidimensional T1rho-, diffusion-filtered and diffusion-ordered NOESY techniques were applied to identify segments of a ligand binding with a protein receptor. These experiments can easily provide intermolecular NOEs of the complex, which are of great significance for characterizing the binding epitopes of a ligand. This information cannot be obtained by high-throughput 1D NMR experiments, used for determining the binding affinity, although multidimensional NMR experiments require more experiment time. The present results indicate that the current experiments using T1rho- and diffusion-edited techniques are very suitable for identifying segments of a ligand binding with a protein receptor in the drug discovery process.     

蛋白质分子与配体结合过程构象变化的研究

蛋白质分子与配体的作用模式主要有直接的环区结合及铰链式结合两种方式。针对这两种不同的作用方式,我们提出采用不同的策略进行结合过程的构象研究。对于直接的环区结合模式,通过建立环区主链构象库,来实现蛋白质环区与配体的准柔性对接,并以链霉抗生物素蛋白体系为例对构象库建立的可行性进行了验证计算。对铰链结合方式,采用分步对接的方法进行计算,并具体应用于HIV蛋白酶与其小分子配体的结合过程。计算结果表明,这两种处理方法分别能较好地模拟不同类型的蛋白质与配体结合的的构象变化。     

Computational study of ligand binding in lipid transfer proteins: Structures,interfaces, and free energies of protein‐lipid complexes

Plant nonspecific lipid transfer proteins (nsLTPs) bind a wide variety of lipids, which allows them to perform disparate functions. Recent reports on their multifunctionality in plant growth processes have posed new questions on the versatile binding abilities of these proteins. The lack of binding specificity has been customarily explained in qualitative terms on the basis of a supposed structural flexibility and nonspecificity of hydrophobic protein‐ligand interactions. We present here a computational study of protein‐ligand complexes formed between five nsLTPs and seven lipids bound in two different ways in every receptor protein. After optimizing geometries in molecular dynamics calculations, we computed Poisson‐Boltzmann electrostatic potentials, solvation energies, properties of the protein‐ligand interfaces, and estimates of binding free energies of the resulting complexes. Our results provide the first quantitative information on the ligand abilities of nsLTPs, shed new light into protein‐lipid interactions, and reveal new features which supplement commonly held assumptions on their lack of binding specificity. © 2012 Wiley Periodicals, Inc.     

Application of NMR screening techniques for observing ligand binding with a protein receptor

Water ligand observed via gradient spectroscopy (WaterLOGSY), saturation transfer difference and NOE pumping NMR techniques were used to identify ligand binding with a receptor. Although these experiments were originally designed to observe ligands in complexes, their application is limited by the affinity of ligands towards target molecules. Here the improved WaterLOGSY pulse sequence was developed by incorporating the double pulsed field gradient spin-echo and gradient-tailored excitation WATERGATE sequences. The efficiency of these ligand-observed NMR screening techniques was investigated using the ribonuclease T1-inhibitor system.     

Nonlinearly additive forces in multivalent ligand binding to a single protein revealed with force spectroscopy

We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between concanavalin A (ConA) and alpha-D-mannose but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 68, and 85 pN at a loading rate of 650 +/- 25 pN/s, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and nonspecific binding. We analyze the binding configuration (i.e., serial vs parallel connections) through fitting the polymer stretching data with modified wormlike chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.     

Investigation of the copper binding site and the role of histidine as a ligand in riboflavin binding protein

Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 A, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O 3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.     

Synthesis and magnetic properties of a highly conducting neutral nickel complex with a highly conjugated tetrathiafulvalenedithiolate ligand

A neutral nickel complex with the extended tetrathiafulvalene-4,5-dithiolate ligand benzotetrathiafulvalenedithiolate (btdt2-) is synthesized and characterized. From the structural analysis, the neighboring molecules in this complex are stacked in a different way compared to the previously reported single-component metal [Ni(tmdt)2]. The computational studies confirm that the difference in molecular packing results in the variance of conductivity.     

pH-dependent protein conformational changes in albumin:gold nanoparticle bioconjugates: a spectroscopic study

The conformational changes of bovine serum albumin (BSA) in the albumin:gold nanoparticle bioconjugates were investigated in detail by various spectroscopic techniques including UV-vis absorption, fluorescence, circular dichroism, and Fourier transform infrared spectroscopies. Our studies suggested that albumin in the bioconjugates that was prepared by the common adsorption method underwent substantial conformational changes at both secondary and tertiary structure levels. BSA was found to adopt a more flexible conformational state on the boundary surface of gold nanoparticles as a result of the conformational changes in the bioconjugates. The conformational changes at pH 3.8, 7.0, and 9.0, which corresponded to different isomeric forms of albumin, were investigated, respectively, to probe the pH effect on the conformational changes of BSA in the bioconjugates. The results showed that the pH of the medium influenced the changes greatly and that fluorescence and circular dichroism studies further indicated that the changes were larger at higher pH.     

Kinetic stabilization of an oligomeric protein by a single ligand binding event

Protein native state stabilization imposed by small molecule binding is an attractive strategy to prevent the misfolding and misassembly processes associated with amyloid diseases. Transthyretin (TTR) amyloidogenesis requires rate-limiting tetramer dissociation before misassembly of a partially denatured monomer ensues. Selective stabilization of the native TTR tetramer over the dissociative transition state by small molecule binding to both thyroxine binding sites raises the kinetic barrier of tetramer dissociation, preventing amyloidogenesis. Assessing the amyloidogenicity of a TTR tetramer having only one amyloidogenesis inhibitor (I) bound is challenging because the two small molecule binding constants are generally not distinct enough to allow for the exclusive formation of TTR.I in solution to the exclusion of TTR.I(2) and unliganded TTR. Herein, we report a method to tether one fibril formation inhibitor to TTR by disulfide bond formation. Occupancy of only one of the two thyroxine binding sites is sufficient to inhibit tetramer dissociation in 6.0 M urea and amyloidogenesis under acidic conditions by imposing kinetic stabilization on the entire tetramer. The sufficiency of single occupancy for stabilizing the native state of TTR provides the incentive to search for compounds displaying striking negative binding cooperativity (e.g., K(d1) in nanomolar range and K(d2) in the micromolar to millimolar range), enabling lower doses of inhibitor to be employed in the clinic, mitigating potential side effects.     

Alkyne oligomerization mediated by rhodium complexes with a phosphinosulfonamido ligand and isolation and characterization of a rhodacyclopentadiene complex

Treatment of N-tosyl aziridine with KPPh₂ in THF produces Ph₂PCH₂CH₂NTsK (Ts = p-CH₃C₆H₄SO₂). Reaction of Ph₂PCH₂CH₂NTsK with [Rh₂(μ₂-Cl)₂(NBD)₂] (NBD = norbornadiene) and [Rh₂(μ₂-Cl)₂(COD)₂] (COD = 1,5-cyclooctadiene) produces [Rh(NBD)(Ph₂PCH₂CH₂NTs)] and [Rh(COD)(Ph₂PCH₂CH₂NTs)] (4), respectively. Reaction of Ph₂PCH₂CH₂NTsK with [Ir₂(μ₂-Cl)₂(COD)₂] gives [Ir(COD)(Ph₂PCH₂CH₂NTs)]. Complex 4 is catalytically active for polymerization of arylalkynes and for cyclotrimerization of HCCCOR (R = OEt, Me). The novel metallacycle [Rh(C(CO₂Et)CHC(CO₂Et)CH)(CH(CO₂Et)CCCCO₂Et)(Ph₂PCH₂CH₂NHTs)₂] was isolated from the reaction of 4 with ethyl propiolate. The metallacycle is catalytically active for cyclotrimerization of ethyl propiolate.     

Computational and experimental investigations of mono-septanoside binding by Concanavalin A: correlation of ligand stereochemistry to enthalpies of binding

Structure-energy relationships for a small group of pyranose and septanose mono-saccharide ligands are developed for binding to Concanavalin A (ConA). The affinity of ConA for methyl "manno"β-septanoside 7 was found to be higher than any of the previously reported mono-septanoside ligands. Isothermal titration calorimetry (ITC) in conjunction with docking simulations and quantum mechanics/molecular mechanics (QM/MM) modeling established the specific role of binding enthalpy in the structure-energy relations of ConA bound to natural mono-saccharides and unnatural mono-septanosides. An important aspect in the differential binding among ligands is the deformation energy required to reorganize internal hydroxyl groups upon binding of the ligand to ConA.     

An efficient use of the WATERGATE W5 sequence for observing a ligand binding with a protein receptor

An efficient pulse sequence for observing a ligand binding with a receptor has been developed by incorporating the WATERGATE W5 sequence. In the conventional water ligand observed via gradient spectroscopy (WaterLOGSY) techniques, the water resonance is selectively excited using, e.g. the double-pulsed field gradient spin-echo (DPFGSE) sequence at the initial portion of pulse sequence. In the current version, the modified WATERGATE W5 sequence is incorporated at the initial portion of the pulse sequence, and the resonance at the water frequency can be selectively reserved by the modified WATERGATE W5 sequence. The efficiency of ligand-observed NMR screening techniques has been demonstrated using the human serum albumin (HSA)-tryptophan complex.     

pH-dependent self-assembly of copper(II) complexes with a new imidazole-containing polyamine ligand: Synthesis,structure and magnetic property

Four copper(II) complexes were synthesized by reactions of new imidazole-containing polyamine ligand N¹-(2-aminoethyl)-N¹-(1H-imidazol-4-ylmethyl)-ethane-1,2-diamine (HL) with Cu(ClO₄)₂ · 6H₂O under different pH and their structures were characterized by X-ray crystallography. Interestingly, the complexes have diverse structures from protonated ligand [H₃(HL)][CuCl₄] · Cl (1), dinuclear [Cu₂(HL)₂Cl](ClO₄)₃ · H₂O (2), one-dimensional chain polynuclear {[Cu(L)](ClO₄)}_n (3) to cyclic-tetranuclear [Cu₄(L)₄](ClO₄)₄ · 3CH₃CN (4) coordination compounds by varying reaction pH from acidic to basic. The results indicate that the reaction pH has great impact on the formation and structure of the complexes. The magnetic measurements show that there are antiferromagnetic interactions between the Cu(II) centers with g = 2.09, J = −39.0 cm⁻¹ and g = 2.17, J = −36.8 cm⁻¹ for 3 and 4, respectively.     

A fluorescence-based assay for nanogram quantification of proteins using a protein binding ligand

Fluorescence emission has been investigated in the context of estimation of proteins at nanogram levels. A Schiff base ligand with donor-acceptor substituents has been utilized as a fluorescent probe. The potency of this ligand is that it possesses the binding sites for both hydrophobic as well as hydrophilic groups in the proteins. The fluorescence emission of the probe was enhanced in the presence of nanogram levels of protein, which clearly signifies that even the least concentration of the protein is sufficient to perturb the environment around the probe. We demonstrate here that the fluorescence characteristic of the probe can be utilized to estimate even nanogram levels (66 ng-1 microgram mL(-1)) of protein. The major limitation of the currently available standard methods is the range of protein estimation, which terminates at microgram level and the interference due to the specificity of the amino acids, which vary from proteins to proteins. This fluorescence emission-based method is free from interference from any type of buffers, ionic strength of the medium and any specific amino acid residue and is a simple, rapid, single-step, sensitive method of estimation which can be applied to different classes of proteins.     

Mass spectrometry and NMR analysis of ligand binding by human liver fatty acid binding protein

Human liver fatty acid binding protein (hL‐FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a ‘head‐in’ or ‘head‐out’ orientation of the carboxylate end. Electrospray‐ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head‐out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of difference NOE-pumping NMR technique and cold-spray ionization mass spectrometry to identify a ligand binding with a protein receptor.

A difference diffusion-based NMR technique and cold-spray ionization mass spectrometry were employed as a solution-based approach for identifying a ligand binding with a protein receptor. The difference diffusion-based NMR technique, called difference NOE-pumping, can directly detect the ligand interacting with a protein receptor. This technique uses a simple pulse sequence and the diffusion filter can easily be optimized. The cold-spray ionization mass spectrometry (CSI-MS), a variant of electrospray ionization mass spectrometry (ESI-MS) operating at low temperature, has been applied to detect the ligand-receptor complex. The efficiency of these techniques for identifying binding ligands is demonstrated with the human serum albumin (HSA)-drug system.