A fractal analysis of protein to DNA binding kinetics using biosensors

A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.     

A multi-step approach to time series analysis and gene expression clustering

MOTIVATION: The huge growth in gene expression data calls for the implementation of automatic tools for data processing and interpretation. RESULTS: We present a new and comprehensive machine learning data mining framework consisting in a non-linear PCA neural network for feature extraction, and probabilistic principal surfaces combined with an agglomerative approach based on Negentropy aimed at clustering gene microarray data. The method, which provides a user-friendly visualization interface, can work on noisy data with missing points and represents an automatic procedure to get, with no a priori assumptions, the number of clusters present in the data. Cell-cycle dataset and a detailed analysis confirm the biological nature of the most significant clusters. AVAILABILITY: The software described here is a subpackage part of the ASTRONEURAL package and is available upon request from the corresponding author. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.     

Measurement of the affinity of melittin for zwitterionic and anionic membranes using immobilized lipid biosensors.

The binding of melittin to zwitterionic dimyristyphosphatidylcholine (DMPC) and anionic dimyristylphosphatidylglycerol (DMPG) was analysed using two different immobilized model membrane systems. The first system used surface plasmon resonance (SPR), which monitors the real-time binding of peptides to an immobilized hybrid bilayer. SPR experiments reflected a stronger binding of melittin for DMPG than for DMPC, while kinetic analysis suggested the existence of at least two distinct binding steps. The second lipid biosensor system involved an immobilized phospholipid monolayer covalently attached to a microporous silica surface. The binding of melittin to the immobilized monolayer was then monitored using dynamic elution chromatography with varied methanol concentrations to analyse the binding of melittin to DMPC and DMPG. The nonlinear binding behaviour observed for melittin with the phosphatidylcholine (PC) and phosphatidylglycerol (PG) monolayers compared with the linear retention plots and Gaussian peak shapes observed for the control molecule demonstrated that melittin undergoes significant conformational and orientational changes upon binding to the immobilized PC and PG ligands. The dependence of log k' on per cent methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while other forces, presumably electrostatic in nature, also made a contribution to the affinity of the peptides for the lipid monolayer, particularly at lower temperatures. The complementary use of these two lipid biosensors thus allows the role of hydrophobic and electrostatic forces in peptide-membrane interactions to be studied.     

Mannose glycoconjugates functionalized at positions 1 and 6. Binding analysis to DC-SIGN using biosensors

The design of glycoconjugates to allow the generation of multivalent ligands capable of interacting with the receptor DC-SIGN is a topic of high interest due to the role played by this lectin in pathogen infections. Mannose, a ligand of this lectin, could be conjugated at two different positions, 1 and 6, not implicated in the binding process. We have prepared mannose conjugates at these two positions with a long spacer to allow their attachment to a biosensor chip surface. Analysis of the interaction between these surfaces and the tetravalent extracellular domain (ECD) of DC-SIGN by SPR biosensor has demonstrated that both positions are available for this conjugation without affecting the protein binding process. These results emphasize the possibility to conjugate mannose at position 6, allowing the incorporation of hydrophobic groups at the anomeric position to interact with hydrophobic residues in the carbohydrate recognition domain of DC-SIGN, increasing binding affinities. This fact is relevant for the future design of new ligands and the corresponding multivalent systems for DC-SIGN.     

A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental and biomedical effects

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ERs) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions, and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate and deleterious to health signaling) may be better controlled. The fractal approach is applied to analyte–ER interaction data available in the literature. The units for the different parameters (rate coefficients and affinities) in fractal-type kinetics are different from those obtained in classical kinetics. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D_f) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. This has biomedical and environmental implications in that the dissociation (and the binding) rate coefficient may be used to alleviate (deleterious effects) or enhance (beneficial effects) by selective modulation of the surface. The affinity values obtained in the analysis are consistent with the numbers required to (a) promote signaling between the correct analyte and the estrogen receptor, and (b) minimize the signaling between xenoestrogens and the estrogen receptor.     

Solubilization of high affinity G-protein-coupled serotonin 1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration

The serotonin 1A (5-HT 1A ) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT 1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT 1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT 1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT 1A receptor in particular, and G-protein-coupled receptors in general.     

Solubilization of high affinity G-protein-coupled serotonin1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration

The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT1A receptor in particular, and G-protein-coupled receptors in general.     

Urbanization and time of arrival of common birds at garden feeding stations

Capsule Birds appeared at garden feeding stations later in the morning in urban areas than in rural areas during winter.     

Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors

We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.     

TREES SIFTER 1.0: an approximate method to estimate the time to the most recent common ancestor of a sample of DNA sequences

trees sifter 1.0 implements an approximate method to estimate the time to the most recent common ancestor (TMRCA) of a set of DNA sequences, using population evolution modelling. In essence, the program simulates genealogies with a user‐defined model of coalescence of lineages, and then compares each simulated genealogy to the genealogy inferred from the real data, through two summary statistics: (i) the number of mutations on the genealogy (M_n), and (ii) the number of different sequence types (alleles) observed (K_n). The simulated genealogies are then submitted to a rejection algorithm that keeps only those that are the most likely to have generated the observed sequence data. At the end of the process, the accepted genealogies can be used to estimate the posterior probability distribution of the TMRCA.     

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.     

Autologous transfusion of erythrocytes with high or low concentration of 2,3-diphosphoglycerate. Survival rate and time and hemoglobin-oxygen affinity]

1. In human erythrocytes the 2.3 DPG concentration was increased three to fourfold of the norm as IPP re-suspension by an incubation time of four hours at 37 degrees C or as ACD-AG blood was lowered below 20% of the norm respectively. After an autologous transfusion the 24 hours' surviving rate and the apparent half survival time of cells as well as the affinity of haemoglobin to oxygen in the total blood were measured. 2. The 24 hours' surviving rate for fresh erythrocytes with increased 2.3 DPG and ATP concentration amounts to 73% and the apparent half survival time amounts to 6 days. If erythrocytes are stored for four weeks as IPP resuspension at 4 degrees C, the 24 hours' surviving rate is 59%. Erythrocytes from fresh ACD-AG blood with lowered 2.3 DPG and a normal ATP concentration have a 24 hours' surviving time of 85% and an apparent half survival time of 24 days. 3. After autologous transfusion of 400 ml of erythrocytes with increased 2.3 DPG concentration the P50 value of the total blood will increase by 3 mm of Hg, after administering 400 ml of erythrocytes with lowered 2.3 DPG concentration it will fall by 1.8 mm of Hg. 4. The findings are discussed in connection with the significance of the changes of affinity of haemoglobin to oxygen produced by 2.3 DPG for the oxygen supply of tissues and under the aspect of using stored blood with increased 2.3 DPG concentration for practical purposes.     

Determination of elasticities, concentration and flux control coefficients from transient metabolite data using linlog kinetics

This paper presents a practical approach to estimate the kinetic parameters of a metabolic network from in vivo kinetics experiments. This method is based on the linlog kinetics format (Visser and Heijnen, 2003, Metab. Eng. 5(3), 164-176; Wu et al., 2004, Eur. J. Biochem. 271, 3348-3359), of which the kinetic parameters, called elasticities, are estimated by an iterative linear optimization followed by non-linear optimization, from transient metabolite concentration data which are directly obtainable from rapid pulse experiments. In this way, not only the parameters are estimated but also a full kinetic model, based on linlog kinetics, is developed. The obtained elasticities also allow immediate calculation of all control coefficients. As an in silico case study, the estimation of elasticities of a linear pathway is presented. The method is shown to be able to estimate the elasticities quite accurately and to be robust toward errors in the metabolite data originating from sampling and measurement inaccuracy. The method allows experimental redesign to get more accurate estimated parameters and accommodates various types of experimentally applied disturbances in the pathway: changes in independent metabolites, dependent metabolites or enzyme levels/activities.     

Microfabricated modules for sample handling, sample concentration and flow mixing: application to protein analysis by tandem mass spectrometry

The comprehensive analysis of biological systems requires a combination of genomic and proteomic efforts. The large-scale application of current genomic technologies provides complete genomic DNA sequences, sequence tags for expressed genes (EST's), and quantitative profiles of expressed genes at the mRNA level. In contrast, protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the proteins expressed by a tissue or cell. The sensitivity of protein analysis technology is primarily limited by the loss of analytes, due to adsorption to surfaces, and sample contamination during handling. Here we summarize our work on the development and use of microfabricated fluidic systems for the manipulation of minute amounts of peptides and delivery to an electrospray ionization tandem mass spectrometer. New data are also presented that further demonstrate the potential of these novel approaches. Specifically, we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification. We also describe the use of a microfabricated module for the generation of solvent gradients at nl/min flow rates for gradient chromatography-tandem mass spectrometry. The use of microfabricated fluidic systems reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces. The ability to assemble dedicated modular systems and to operate them automatically makes the use of microfabricated systems attractive for the sensitive and large-scale analysis of proteins.