Binding and reversibility of Thermobifida fusca Cel5A,Cel6B,and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5 degrees C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in "region I" although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme-substrate complex within the active site of the CDs.     

The effect of multiple binding modes on empirical modeling of ligand docking to proteins.

A popular approach to the computational modeling of ligand/receptor interactions is to use an empirical free energy like model with adjustable parameters. Parameters are learned from one set of complexes, then used to predict another set. To improve these empirical methods requires an independent way to study their inherent errors. We introduce a toy model of ligand/receptor binding as a workbench for testing such errors. We study the errors incurred from the two state binding assumption--the assumption that a ligand is either bound in one orientation, or unbound. We find that the two state assumption can cause large errors in free energy predictions, but it does not affect rank order predictions significantly. We show that fitting parameters using data from high affinity ligands can reduce two state errors; so can using more physical models that do not use the two state assumption. We also find that when using two state models to predict free energies, errors are more severe on high affinity ligands than low affinity ligands. And we show that two state errors can be diagnosed by systematically adding new binding modes when predicting free energies: if predictions worsen as the modes are added, then the two state assumption in the fitting step may be at fault.     

Equilibrium competition binding assay: inhibition mechanism from a single dose response

Inhibition of a receptor by a small-molecule compound in many cases is achieved via a competitive, uncompetitive or non-competitive mechanism. The receptor-inhibitor interaction is often probed through the displacement of a ligand in an equilibrium competition binding experiment. The previous solutions to receptor inhibition mechanisms were borrowed from steady-state enzyme inhibition mechanisms. The inhibition mechanism is determined by a visual inspection or a global fit of ligand dose response curves at a series of inhibitor concentrations. However these solutions only apply to situations when both the ligand and the inhibitor are not significantly depleted by the receptor. In most published equilibrium receptor binding studies, only the relative potency of the inhibitor is calculated. Ranking inhibitors tested under differing experimental conditions is often not possible. In the current paper, we offer exact mathematical solutions to uncompetitive and non-competitive inhibition, and demonstrate that in most cases both the inhibition mechanism and absolute potency of an inhibitor can be simultaneously determined from a single dose response of the inhibitor at a fixed concentration of the ligand. Therefore, an equilibrium competition assay provides a quick and facile method to determine the inhibition mechanism of a large number of inhibitors. The theory herein described is applicable to equilibrium competition binding experiments such as radioligand assays and fluorescence polarization assays.     

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.     

Number of receptor sites from Scatchard and Klotz graphs: complementary approaches

Estimates of number of receptor sites and evaluation of the complexity of the binding process require collection of a spectrum of binding measurements and selection of a theoretical model to fit the experimental data. The appropriateness of the measurements and of the model can be visually judged on graphic displays of the model-data fitting curves in Scatchard and semilogarithmic coordinates. This approach is helpful for detecting the two types of errors most frequently found in reports of binding studies: (1) underestimating the number of binding sites, and (2) failure to recognize the complexity of the binding process. While the former is readily recognizable on semilogarithmic but not on Scatchard plots of the model fitting the data, the latter might not be apparent on either plot. Collection of extensive measurements over a wide range of ligand concentrations with graphic display of the model-data fitting curves in Scatchard and semilogarithmic coordinates should be used to recognize and prevent both errors.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

FTMove: A Web Server for Detection and Analysis of Cryptic and Allosteric Binding Sites by Mapping Multiple Protein Structures

Protein mapping distributes many copies of different molecular probes on the surface of a target protein in order to determine binding hot spots, regions that are highly preferable for ligand binding. While mapping of X-ray structures by the FTMap server is inherently static, this limitation can be overcome by the simultaneous analysis of multiple structures of the protein. FTMove is an automated web server that implements this approach. From the input of a target protein, by PDB code, the server identifies all structures of the protein available in the PDB, runs mapping on them, and combines the results to form binding hot spots and binding sites. The user may also upload their own protein structures, bypassing the PDB search for similar structures. Output of the server consists of the consensus binding sites and the individual mapping results for each structure - including the number of probes located in each binding site, for each structure. This level of detail allows the users to investigate how the strength of a binding site relates to the protein conformation, other binding sites, and the presence of ligands or mutations. In addition, the structures are clustered on the basis of their binding properties. The use of FTMove is demonstrated by application to 22 proteins with known allosteric binding sites; the orthosteric and allosteric binding sites were identified in all but one case, and the sites were typically ranked among the top five. The FTMove server is publicly available at https://ftmove.bu.edu.     

Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions

C-reactive protein (CRP) is a clinically important classic acute phase pentameric protein. It is thought to play an important role in immunomodulation. Earlier reports convincingly demonstrated that human CRP is differentially glycosylated in different pathological conditions. Although CRP is considered to be a clinically important molecule, changes in binding characteristics with appropriate ligands with respect to glycosylation remain unexplored. In an effort to demonstrate that these glycosylated molecular variants are capable of modulating their binding activity with different ligands, CRPs were affinity purified from six different clinical samples. Variable amounts of linkage-specific sialic acid derivatives were found in these CRPs with varying tryptophan contents. Differential binding patterns with antibodies against human CRP, human IgG, and other ligands like fibronectin, fetuin, and asialofetuin indicated that the purified CRPs differed significantly in their lectin-like interactions. Thus, we have convincingly demonstrated that differentially induced CRPs exhibited variable binding characteristics. These results may have far reaching practical applications for understanding acute phase responses. Published in 2004..     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

In vitro and in vivo regulation of chJoroplast glyceraldehyde-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. III. The molecular basis of the aggregation phenomenon: chloroplast glyceraldehyde-3-phosphate dehydrogenase as an ambiquitous enzyme

The nature of the aggregated form of chloroplast glyceraldehyde-3-phosphate dehydrogenase isozymes (GPD, EC 1.2.1.13) from Chenopodium rubrum leaves was investigated. After disaggregation of the isozymes in NADP ⁺ buffer, and resuspension of the disaggregated isozymes in NAD⁺ buffer, complete reaggregation could only be achieved by remixing the enzyme with a high molecular weight fraction, from which the isozymes had dissociated during the NADP⁺ filtration. After separation of the isozymes by inverse ammonium sulphate gradient solubilization, spontaneous extensive reaggregation of each isozyme was observed in NAD⁺ buffer. The high molecular weight material consisted of ribonucleoprotein, and RNase treatment impaired its ability to promote reaggregation of chloroplast GPD. It is proposed that pyridine nucleotide-controlled aggregation and binding to ribonucleoprotein in vitro are artifacts which reflect an in situ binding to cellular components. Since uncontrolled NAD⁺-linked activities of the bifunctional isozymes in the chloroplast would lead to an equalization of the NAD ⁺ and NADP ⁺ redox couples, it is suggested that the reversible binding of the isozymes forms the basis of a regulatory system in vivo.     

On the interpretation of binding isotherms in complex biological systems: Apparent homogeneity of some heterogeneous systems

A simple treatment of the effect of site heterogeneity upon binding isotherms is presented, which is applicable to the analysis of data obtained from measurements of hormone, drug, or lectin binding to membranes and cell surfaces. Using this treatment, isotherms corresponding to various distributions of binding constants have been fitted to examples of experimental binding data ordinarily interpreted in the context of a homogeneous binding site model. It is found that these data do not permit one to exclude the alternate possibility of a broad distribution of the binding constant $\text{K}$. If a homogeneous binding site model can be satisfactorily fitted to the data, it is probable that the value of $\text{K}$ obtained by this procedure is equal or nearly equal to the number average value of $\text{K}$ in the actual (unknown) distribution.     

Hydrogen bonding motifs of protein side chains: Descriptions of binding of arginine and amide groups

The modes of hydrogen bonding of arginine, asparagine, and glutamine side chains and of urea have been examined in small-molecule crystal structures in the Cambridge Structural Database and in crystal structures of protein-nucleic acid and protein-protein complexes. Analysis of the hydrogen bonding patterns of each by graph-set theory shows three patterns of rings (R) with one or two hydrogen bond acceptors and two donors and with eight, nine, or six atoms in the ring, designated R2 2(8), R2 2(9), and R1 2(6). These three patterns are found for arginine-like groups and for urea, whereas only the first two patterns Rl(8) and R²(9) are found for asparagine- and glutamine-like groups. In each case, the entire system is planar within 0.7 Å or less. On the other hand, in macromolecular crystal structures, the hydrogen bonding patterns in protein-nucleic acid complexes between the nucleic acid base and the protein are all R2 2(9), whereas hydrogen bonding between Watson-Crick-like pairs of nucleic acid bases is R2 2(8). These two hydrogen bonding arrangements [R2 2(9) and R2 2(8)] are predetermined by the nature of the groups available for hydrogen bonding. The third motif identified, R1 2(6), involves hydrogen bonds that are less linear than in the other two motifs and is found in proteins.     

The binding of cationic surfactants by DNA

Isotherms for the binding of dodecyltrimethylammonium (DTA⁺) and tetradecyltrimethylammonium (TTA⁺) ions by DNA in aqueous solution at 30°C are reported. The binding isotherms were determined using a potentiometric technique with cationic surfactant-selective electrodes. The DNA concentrations used are 5 × 10^?4 and 10^?3 equiv./kg, Surfactant concentrations varying from 3 × 10^?6M to the critical micelle concentration. The influence of added NaCl (0.01 M) on the binding process is studied. The binding process is shown to be highly cooperative. Applying the binding theory of Schwarz and of Satake and Yang, binding constants and cooperativity parameters can be calculated. The binding constant K is found to be 1.2kT larger for TTA⁺ than for DTA⁺ in salt-free solution, and 1.4kT larger for TTA⁺ than for DTA⁺ in 0.01 M NaCl. The cooperativity parameter u is about 1.4kT larger for TTA⁺ in salt-free solution and 1.2kT larger in 0.01 M NaCl. It is concluded that the hydrophobic part of the bound surfactant is not completely immersed in the hydrophobic DNA core, but also interacts with other surfactant molecules. This situation is compared to the case of micelle formation.     

胰瘘发生假说到捆绑式胰肠吻合的临床运用

为避免术后胰瘘的发生,在过去的100年间提出了多种胰肠吻合重建方式,但哪种方法最好一直备受争议.胰肠吻合后吻合针距间存在可能的间隙,这个问题或许成为胰肠吻合口瘘的爆发点,而且存在贯穿浆肌层缝合的胰腺表面外露的吻合针眼,如果缝针贯穿胰管小分支,胰液可能从针眼流出.这一假说构成了捆绑式胰肠吻合的基础.捆绑式胰肠吻合是一种安全、有效的预防术后胰瘘的吻合方式.     

Diffusivity of Cu(2+) in calcium alginate gel beads

A linear absorption model (LAM) is used to describe the process of metal binding to spherically shaped biopolymers particles. The LAM was solved using a numerical algorithm which calculates diffusivities of metal ion in biopolymer gels. It assumes attainment of rapid metal-biopolymer binding equilibrium accompanied by rate limiting diffusion of the metal ions through the gel. The model was tested using batch experiments in which copper (Cu(2+)) binding with calcium alginate beads was investigated. Biopolymer density in the beads was varied between 2% and 5%. The diffusion coefficient of Cu(2+) calculated from the LAM ranged from 1.19 x 10(-9) to 1.48 x 10(-9) m(2) s(-1) (average 1.31 +/- 0.21 x 10(-9) m(2) s(-1)), independent of biopolymer density. The LAM has theoretical advantages over the shrinking core model (shell progressive model). The latter calculated an unreasonable exponential increase in the diffusion coefficient as density of alginate polymer in the bead increased. (c) 1993 John Wiley & Sons, Inc.     

DNA与蛋白质结合的荧光测定

构建了插入λ阻抑蛋白（Ｒｅｐ）的操纵基因（ＯＲ）和ＢｇｌⅡ识别位点的ＰＢＲ３２２重组质粒。阻抑蛋白与该质粒的相互作用可用ＢｇｌⅡ对它的水解作用引起的ＥＢ荧光变化来研究。在Ｅ．ｃｏｌｉ中表达的Ｒｅｐ表现了与该重组质粒结合的活力。核苷酸序列具精确二重对称性的ＯＲ（ＯＲｃｏｎｓ）对Ｒｅｐ的亲和力比天然的ＯＲ１小。     

木聚糖酶碳水化合物结合结构域研究进展

木聚糖酶含有催化活性结构域,有时还含有非催化活性结构域,促进酶与底物结合,特别是与不溶性底物的结合及降解,称为碳水化合物结合结构域(CBM),它们在木聚糖降解过程中有重要作用。以下从CBM来源,所属家族类型、对不溶性底物结合特性、与底物结合的特定氨基酸、与催化结构域间的连接肽、特别是对影响木聚糖酶稳定性的5个方面进行了综述,说明CBM对木聚糖酶性质有很大影响。自然界中碳水化合物结构复杂、难以降解,所以认识CBM相关性质对研究其与木聚糖酶的协同作用、提高木聚糖酶活性有重要意义,并根据CBM属性用于改造木聚糖酶相关性质进行了展望。     

A method for determining binding kinetics applied to thiamine-binding protein

A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.