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Recent experiments have suggested that enzymes and other small molecules chemotax toward their substrates. However, the physical forces driving this chemotaxis are currently debated. In this work, we consider a simple thermodynamic theory for molecular chemotaxis that is based on the McMillan-Mayer theory of dilute solutions and Schellman's theory for macromolecular binding. Even in the absence of direct interactions, the chemical binding equilibrium introduces a coupling term into the relevant free energy, which then reduces the chemical potential of both enzymes and their substrates. Assuming a local thermodynamic equilibrium, this binding contribution to the chemical potential generates an effective thermodynamic force that promotes chemotaxis by driving each solute toward its binding partner. Our numerical simulations demonstrate that, although small, this thermodynamic force is qualitatively consistent with several experimental studies. Thus, our study may provide additional insight into the role of the thermodynamic binding free energy for molecular chemotaxis.

Computational structural prediction of macromolecular interactions is a fundamental tool toward the global understanding of cellular processes. The Critical Assessment of PRediction of Interactions (CAPRI) community-wide experiment provides excellent opportunities for blind testing computational docking methods and includes original targets, thus widening the range of docking applications. Our participation in CAPRI rounds 38 to 45 enabled us to expand the way we include evolutionary information in structural predictions beyond our standard free docking InterEvDock pipeline. InterEvDock integrates a coarse-grained potential that accounts for interface coevolution based on joint multiple sequence alignments of two protein partners (co-alignments). However, even though such co-alignments could be built for none of the CAPRI targets in rounds 38 to 45, including host-pathogen and protein-oligosaccharide complexes and a redesigned interface, we identified multiple strategies that can be used to incorporate evolutionary constraints, which helped us to identify the most likely macromolecular binding modes. These strategies include template-based modeling where only local adjustments should be applied when query-template sequence identity is above 30% and larger perturbations are needed below this threshold; covariation-based structure prediction for individual protein partners; and the identification of evolutionarily conserved and structurally recurrent anchoring interface motifs. Overall, we submitted correct predictions among the top 5 models for 12 out of 19 interface challenges, including four High- and five Medium-quality predictions. Our top 20 models included correct predictions for three out of the five targets we missed in the top 5, including two targets for which misleading biological data led us to downgrade correct free docking models.

Binding of macromolecules to surfaces, or to surface-attached binding partners, is usually described by the classical Langmuir model, which does not include interaction between incoming and adsorbed molecules or between adsorbed molecules.The present study introduces the “Surfint” model, including such interactions. Instead of the exponential binding behaviour of the Langmuir model, the Surfint model has tanh binding equations, as confirmed by a random sequential adsorption (RSA) computer simulation.For high binding affinity, sorption kinetics become diffusion-limited as described by the existing unstirred-layer model “Unstir”, for which we present the exact analytical solution of its binding equations expressed in Lambert W-functions.Low-affinity binding of thrombin on heparin, and high-affinity binding of prothrombin on phospholipid vesicles, were measured by ellipsometry and were best described by the Surfint and Unstir models, respectively.

This review highlights advances in biochemical and medical applications of cage metal complexes (clathrochelates) and related polyhedral compounds as rigid scaffolds for macromolecular binding (topological drugs), in radiation therapy and diagnostics, and as paramagnetic probes for NMR and MRI applications.

Accurate calculation of important thermodynamic properties, such as macromolecular binding free energies, is one of the principal goals of molecular dynamics simulations. However, single long simulation frequently produces incorrectly converged quantitative results due to inadequate sampling of conformational space in a feasible wall-clock time. Multiple short (ensemble) simulations have been shown to explore conformational space more effectively than single long simulations, but the two methods have not yet been thermodynamically compared. Here we show that, for end-state binding free energy determination methods, ensemble simulations exhibit significantly enhanced thermodynamic sampling over single long simulations and result in accurate and converged relative binding free energies that are reproducible to within 0.5 kcal/mol. Completely correct ranking is obtained for six HIV-1 protease variants bound to lopinavir with a correlation coefficient of 0.89 and a mean relative deviation from experiment of 0.9 kcal/mol. Multidrug resistance to lopinavir is enthalpically driven and increases through a decrease in the protein-ligand van der Waals interaction, principally due to the V82A/I84V mutation, and an increase in net electrostatic repulsion due to water-mediated disruption of protein-ligand interactions in the catalytic region. Furthermore, we correctly rank, to within 1 kcal/mol of experiment, the substantially increased chemical potency of lopinavir binding to the wild-type protease compared to saquinavir and show that lopinavir takes advantage of a decreased net electrostatic repulsion to confer enhanced binding. Our approach is dependent on the combined use of petascale computing resources and on an automated simulation workflow to attain the required level of sampling and turn around time to obtain the results, which can be as little as three days. This level of performance promotes integration of such methodology with clinical decision support systems for the optimization of patient-specific therapy.

Previous kinetic investigations have suggested that the process of the specific complex formation goes through intermediate states called target searches or encounter complexes, and the presence of such intermediates greatly accelerates the overall process. However, current biophysics has not provided adequate physical pictures about such intermediates. We have developed new NMR methods that permit investigations of structural and dynamics aspects of the transient, low-population intermediates in protein-DNA or protein-protein association processes. The methods make use of paramagnetic relaxation enhancement (PRE) as a tool to amplify the information about the minor species.

This review highlights advances in biochemical and medical applications of cage metal complexes (clathrochelates) and related polyhedral compounds as rigid scaffolds for macromolecular binding (topological drugs), in radiation therapy and diagnostics, and as paramagnetic probes for NMR and MRI applications.

We present a method for prediction of functional sites in a set of aligned protein sequences. The method selects sites which are both well conserved and clustered together in space, as inferred from the 3D structures of proteins included in the alignment. We tested the method using 86 alignments from the NCBI CDD database, where the sites of experimentally determined ligand and/or macromolecular interactions are annotated. In agreement with earlier investigations, we found that functional site predictions are most successful when overall background sequence conservation is low, such that sites under evolutionary constraint become apparent. In addition, we found that averaging of conservation values across spatially clustered sites improves predictions under certain conditions: that is, when overall conservation is relatively high and when the site in question involves a large macromolecular binding interface. Under these conditions it is better to look for clusters of conserved sites than to look for particular conserved sites.

The standard framework for calculating the absolute binding free energy of a macromolecular association reaction A + B → AB with an association constant K AB is to equate chemical potentials of the species on the left- and right-hand sides of this reaction and evaluate the chemical potentials from theory. This theory involves (usually hidden) assumptions about what constitutes the bound species, AB, and where the contribution of the solvent appears. We present here an alternative derivation that can be traced back to Bjerrum, in which the expectation value of K AB is obtained directly through the statistical mechanical method of evaluating its ensemble (Boltzmann-weighted) average. The generalized Bjerrum approach more clearly delineates: ( i ) the different contributions to binding; ( ii ) the origin of the much-discussed and somewhat controversial association entropy term; and ( iii ) where the solvent contribution appears. This approach also allows approximations required for practical evaluation of the binding constant in complex macromolecular systems, to be introduced in a well defined way. We provide an example, with application to test cases that illustrate a range of binding behavior.

In the present review we summarize recent work, aimed at a better understanding of the interactions in macromolecule–ligand complexes, performed by means of computational tools such as pseudoreceptor generation, molecular docking, conformational search and energy minimization. While the first approach has been applied when the three-dimensional structural properties of the biological target were unknown, the remaining protocols exploited the knowledge of the overall structure of the involved macromolecules and their active sites. Molecular modeling techniques were used in the cases reported to study and propose macromolecular binding sites and to predict their interactions with bioactive conformers of the ligands.

We present a set of programs, DREAM++ (Docking and Reaction programs using Efficient seArch Methods written in C++), for docking computationally generated ligands into macromolecular binding sites. DREAM++ is composed of three programs: ORIENT++, REACT++ and SEARCH++. The program ORIENT++ positions molecules in a binding site with the DOCK algorithm [1, 2]. Its output can be used as input to REACT++ and SEARCH++. The program REACT++ performs user-specified chemical reactions on a docked molecule, so that reaction products can be evaluated for three dimensional complementarity with the macromolecular site. The program SEARCH++ performs an efficient conformation search on the reaction products using a hybrid backtrack [3, 4] and incremental construction [5, 6] algorithm. We have applied the programs to HIV protease–inhibitor complexes as test systems. We found that we can differentiate high-affinity ligands based on several measures: interaction energies, occupancy of protein subsites and the number of successfully docked conformations for each product. Encouraged by the results in the test case, we applied the programs to propose novel inhibitors of HIV protease. These inhibitors can be generated by organic reactions using commercially available reagents. They are alternatives to the inhibitors synthesized by Glaxo [7, 8].

The effect of additives on micellar aggregation is treated using the same framework developed for macromolecular binding. This procedure, hinging on the pseudo-phase transition model, allows us to evaluate the binding constant of any added substance to a micellar aggregate as well as the number of particles bound per amphiphilic unit. The analysis of published literature data confirms that the model provides a general description of micellar aggregation in the presence of added ingredients, regardless of their ionic or non-ionic nature. Original data from our laboratory are well described by the suggested macromolecular binding approach. The dependence of experimental trends on micellization modifiers is also briefly discussed. Copyright 1998 Academic Press.

The identification of small drug-like compounds that selectively inhibit the function of biological targets has historically been a major focus in the pharmaceutical industry, and in recent years, has generated much interest in academia as well. Drug-like compounds are valuable as chemical genetics tools to probe biological pathways in a reversible, dose- and time-dependent manner for drug target identification. In addition, small molecule compounds can be used to characterize the shape and charge preferences of macromolecular binding sites, for both structure-based and ligand-based drug design. High-throughput screening is the most common experimental method used to identify lead compounds. Because of the cost, time, and resources required for performing high-throughput screening for compound libraries, the use of alternative strategies is necessary for facilitating lead discovery. Virtual screening has been successful in prioritizing large chemical libraries to identify experimentally active compounds, serving as a practical and effective alternative to high-throughput screening. Methodologies used in virtual screening such as molecular docking and scoring have advanced to the point where they can rapidly and accurately identify lead compounds in addition to predicting native binding conformations. This chapter provides instructions on how to perform a virtual screen using freely available tools for structure-based lead discovery.

We have examined the changes in the 31P NMR chemical shifts of ATP in aqueous solution, upon addition of NaCl and LiCl at ambient temperature. NaCl and LiCl cause qualitatively similar downfield changes, although the effect of LiCl is somewhat larger. For a 2:1 mixture of Na-ATP or tris-ATP and MgCl2 at 0 degrees C, separate beta-P peaks are observed for uncomplexed ATP and ATP complexed with Mg(II) at 121.6 MHz. Addition of LiCl slightly shifts the uncomplexed Na-ATP or tris-ATP peak downfield but does not measurably shift the Mg-ATP peak or change relative intensities. Thus, Li behaves more like Na than Mg in its ATP complexation behavior, making this system a poor model for competition between Li and Mg for macromolecular binding.

Summary Reliable determination of protein-protein interaction sites is of critical importance for structure-based design of small molecules modulating protein function through macromolecular interfaces. We present an alignment-free computational method for prediction of protein-protein interface residues. The method ("iPred") is based on a knowledge-based scoring function adapted from the field of protein folding and small molecule docking. Based on a training set of 394 hetero -dimeric proteins iPred achieves sustained accuracy on an external unbound test set. Prediction robustness was assessed from more than 1500 diverse complexes containing homo - and hetero -dimers. The technique does not rely on sequence conservation, so that rapid interface identification is possible even for proteins for which homologs are unknown or lack conserved residue patterns in interface region. Functional "hot-spot" residues are enriched among the predicted interface residues, rendering the method predestined for macromolecular binding site identification and drug design studies aiming at modulating protein-protein interaction that might influence protein function. For a comparative structural model of peptidase HtrA from Helicobacter pylori , we performed mutation studies for predicted hot-spot residues, which were confirmed as functionally relevant for HtrA activity or oligomerization.

This chapter presents an introduction to the kinetic analysis of SPR biosensor data for the determination of affinity and kinetic rate constants of biomolecular interactions between an immobilized and a soluble binding partner. The need to be aware of and critically tests the assumptions underlying the analysis models is emphasized and the consequences for the experimental design are discussed. The two most common sources of deviation in SPR surface binding kinetics from the ideal pseudo-first order binding kinetics of bimolecular reactions are mass transport limitations and the heterogeneity of the surface sites. These problems are intrinsic to the use of a biosensor surface for characterizing interactions. The effect of these factors on the observed binding kinetics, and strategies to account for them are reviewed, both in the context of mathematical data analysis, as well as the design of the experiments and controls.

A novel three-dimensional QSAR technique (HASL) is described. HASL (Hypothetical Active Site Lattice) represents a composite description of a macromolecular binding site obtained from ligand molecule structures and activities. The scope and limitations of the method are discussed by means of its application to in vitro and in vivo data.

A generalization of Lifson’s sequence‐generating function method [J. Chem. Phys. 40, 3705 (1964)] for linear chain molecules is explored. The generalization replaces the various scalar series of statistical weights, used to generate sequence partition functions, by appropriate matrix series. These matrix SGFs permit a more compact treatment of complicated macromolecular binding models than can be achieved through the standard SGF method or by using combinatorial or regular transfer matrix methods. They also lead to appreciable gains in computational speed for such models. Applications of the matrix‐SGF method are presented, including selective binding of ligands to helix–coil junctions in biopolymers and sequence‐selective binding of large ligands on random copolymers.

With the introduction of new instruments and improved sensor chip chemistries, surface plasmon resonance (SPR) is finding new applications for molecular interaction studies. Easy access to high-quality kinetic and thermodynamic data for macromolecular binding events is providing insights into the fundamental mechanisms of molecular recognition. Progress is being made to allow larger-scale interaction studies. In addition, combining SPR with other analytical methods is enabling SPR-based analysis of interaction proteomics.

ARBITER is a Prolog program that extracts assertions about macromolecular binding relationships from biomedical text. We describe the domain knowledge and the underspecified linguistic analyses that support the identification of these predications. After discussing a formal evaluation of ARBITER, we report on its application to 491, 000 MEDLINE® abstracts, during which almost 25, 000 binding relationships suitable for entry into a database of macromolecular function were extracted.

The rate constants for macromolecular association reactions depend on the macromolecules involved and the solution conditions. The largest rate constants are for encounter-limited reactions, where association takes place whenever the associating macromolecules collide. Macromolecules that associate through specific binding sites often have much smaller rate constants. Recently, it has been demonstrated that the rate of association for complementary nucleic acid strands can be made encounter limited by attaching weakly associating, relatively unstructured polymeric domains to the nucleic acids involved. These results have led to a model suggesting that similar domains provide a general means of increasing rates of association for specific macromolecular binding partners, both in vivo and in vitro.

A number of mineral dusts were tested for their ability to catalyze the transformation of benzo(a)pyrene from the microcrystalline state into lipid solution. The findings of Lakowicz and his co-workers, that fibrous dusts were more active than nonfibrous dusts, were confirmed. Macromolecular binding metabolites of BaP were formed in A549 cells to a similar extent whether the BaP was added in solution or adsorbed to fibers; however, the level of water-soluble metabolites was lower in cultures treated with adsorbed hydrocarbon. It was found that asbestos can also inhibit the accumulation of 1-naphthyl glucuronide in cultures treated with 1-naphthol. The significance of this in asbestos pathogenesis is briefly discussed.

DBCPによる生体高分子物質のアルキル化と細胞壊死との関係を20mg/kgから致死量の400mg/kgの14C-DBCPをウィスター系雄ラットに1回経口投与して調査した. 14Cはラット体中でおもに肝臓と腎臓の遠位尿細管部に分布した (20および200mg/kg). これら標的臓器における14Cの形態は生体高分子物質と結合したものが主体を占めており, この結合は投与量 (20-400mg/kg) の増大とともに増加した. 肝臓中のグルタチオン量はDBCPの投与量の増加とともに減少し, グルタチオン量と結合量との間に逆相関性が認められた. 組織内のDBCP残留量も投与量に比例して増加したが, いずれの投与量においても組織内14Cのわずかな部分を占めるにすぎなかった.phenobarbital 前処理は致死量DBCPによる致死作用を高め, 肝および腎臓中での細胞壊死強度, 高分子物質への結合, 尿中への排泄を高めた. またそれら組織中のグルタチオン量の低下を促進したが肝臓中の非結合14C量には影響を与えなかった. SKF-525A前処理は上記のDBCPによる効果をいずれも低減させ, また腎臓中の非結合14C量を増大させた. グルタチオンの同時投与は死亡率と高分子結合を抑制し非結合14C量を増大させた. これらの結果から in vivo における高分子物質との結合は in vitro 実験で先に報告したと同様に microsomo 上で形成されるDBCPの活性代謝中間体によるものであり, またこの高分子物質との結合 (アルキル化) が個体死に結びつく細胞壊死を引き起こしていると考えられた. またグルタチオンは標的臓器中のDBCP活性代謝中間体を不活性化して高分子物質との結合を抑制することによって細胞壊死, 致死作用を軽減していると考えられた.

Rats were exposed by inhalation to 5000 ppm of nonlabeled vinyl chloride (VC) 6 hr/day, 5 days/week for 7 weeks. On the last day of repeated exposure 14 C-labeled VC was used. The fate of the [ 14 C]VC was compared in a group of rats exposed repeatedly to a group exposed simultaneously for a single 6 hr period to 5000 ppm of [ 14 C]VC. The routes and rates of excretion of 14 C activity were the same for the two experimental groups. The activity of microsomal enzymes, as reflected by aniline hydroxylase and p -nitroanisole O -demethylase derived from 9000 g liver supernatants, was essentially the same in rats exposed once or repeatedly or in nonexposed control rats. Covalent binding to hepatic macromolecules was greater in rats repeatedly exposed as compared to those subjected to a single exposure. These results indicate that repeated exposure to VC does not induce its biotransformation. However, the increase in hepatic macromolecular binding indicates that repeated exposure augments the reaction of electrophilic metabolites with macromolecules, and this may be expected to enhance potential toxicity including carcinogenicity.

A theoretical model is derived which describes the creation of new binding sites as a result of ligand binding by a two-site creator on the macromolecule. This model may account for unusual Scatchard plots seen in the literature. In addition, a theoretical model is derived to describe ligand-macromolecular interactions in which pre-existing sites are destroyed as a result of macromolecular binding of ligand. Scatchard plots are presented for each model that show the manifestations of the particular ligand-macromolecular interaction.

Denervated muscles due to lumbar radiculopathy, peroneal axonal neuropathy and hypoglossal neuropathy were studied with magnetic resonance imaging (MRI). Denervated muscle was clearly distinguished from normal muscle by its higher intensity signal. Comparison of the resulting signal intensity of the muscle using various imaging parameters suggests an alteration in water macromolecular binding. These preliminary findings indicate that MRI may be useful in the noninvasive in vivo diagnosis and study of the effects of denervation on muscle.

Macromolecular binding of estradiol in the ampullary and isthmic portions of the rabbit oviduct and the rabbit uterus was examined by in vivo uptake studies, sucrose gradient centrifugation, and binding assays. The studies revealed that the in vivo uptake was similar in the 3 mullerian areas examined. In addition, the estrogen receptor complex from all 3 areas had almost identical sedimentation characteristics in sucrose gradient centrifugation. Finally, the binding assays revealed that the number of estrogen-binding sites and the dissociation constants were similar in the 3 areas studied. These results indicate that the initial interaction of estrogen with the uterus and both portions of the oviduct is identical.

We describe a rapid microscale procedure for blood lead determination by anodic stripping voltammetry. Results correlate well with those obtained by atomic absorption spectroscopy. The procedure involves use of a metal-exchange reagent, which rapidly releases bound lead from its macromolecular binding sites, thus eliminating a long and cumbersome perchloric acid digestion procedure. Reproducibility and analytical recovery of added lead were both excellent. Our results show that anodic stripping voltammetry is a reliable, sensitive micromethod for routine determination of blood lead.

An estrogen binding protein for estradiol-17beta is present in the liver cytosol of female intact and one day oophorectomized rats. The dissociation constant reveals high affinity binding (Kd: 0.69 +/- 0.14 times 10(-10) M). Quantitation of EBP using a dextran-coated charcoal method shows that this specific macromolecular binding is much less than in the rat uterus, but similar to that in DMBA-induced mammary tumors. Sucrose density gradient analysis shows sedimentation at 8-9 S and 4-5 S when compared to bovine serum albumin.

The landscape forming the background of this paper is that of the present physicochemical understanding of the molecular interactions between ligands and receptors and the subsequent cellular events that accompany binding. Our laboratory has been active in studies of peptide conformations in solution (1–3) with the object of connecting these with conformations of the same molecules at macromolecular binding sites. Recently, advances in methods have made it possible to study the more biologically meaningful problem of conformations of ligands at specific macromolecular binding sites, especially by nuclear magnetic resonance spectroscopy (4), and we have turned to developing an exemplary system which would enable us to approach this problem. It is via this route that we became interested in the interactions of haptens with antibodies and of Ab2 with anti-hapten Ab1. In particular, the study of opiate ligands (or haptens)1 and their interactions ligands (or haptens)1 and their interactions with opiate receptors has some real technical advantages, and these will be emphasized in this paper.

The use of a synthetic macromolecular hydrophilous binding material for the extraction of multiple renal calculi (“pyeloform” pyelotomy) is reported. An account of the results of animal studiesin vitro is given and the possibilities of the method in humans are pointed out.

The initial chemical reaction step of a large number of complicated processes in living organisms is the association of a low molecular weight ligand to a specific macromolecular binding site. Among the examples for this observation we find substrate enzyme and hormone receptor reactions as well as metal ion activation of proteins and other functionally important biomacromolecular processes. Frequently, the ligand-binding phase is described in anthropomorphic terms such as recognition, discrimination and selection. The meaningful use of these words is, however, limited if more than just binding is to be expressed.

The reaction of anticoagulation system upon perfusion of humorally isolated (with retained innervation) carotid sinus of a rabbit by alpha-,beta/gamma-, DIP-alpha-thrombin and prethrombin I was studied. DIP-alpha-thrombin without clotting activity was shown to initiate like alpha-thrombin the reflex reaction of anticoagulation system characterized by a sharp increase in non-enzymatic fibrinolysis (by 225%) and total fibrinolytic activity of blood (by 51%). Prethrombin I (thrombin precursor) is also capable of exciting the function of anticoagulation system characterized by an increase in non-enzymatic fibrinolysis (by 82%) and total fibrinolytic activity (by 36%). Furthermore, perfusion of prethrombin I or alpha-thrombin at almost the same molar concentrations resulted in the similar degree of anticoagulation system effector reaction. Reflex response of anticoagulation system was not observed upon perfusion of carotid sinus by beta/gamma-thrombin that has high esterase but little if any clotting activity that appears to be due to molecular changes in the macromolecular binding site region. These data support the suggestion that the effect of anticoagulation system excitation is due to interaction of the macromolecular binding site in the structure of alpha-thrombin with anticoagulation system chemoreceptors.

MENTHOR is a database system for the storage and retrieval of three-dimensional coordinate and charge information on molecules as well as of traditional biological and physical properties. Our molecular graphics system retrieves from MENTHOR structural information in individual molecules and receptor map/macromolecular binding site hypotheses. Substructural searches of MENTHOR are used to find starting coordinates for molecular modeling and traditional database searches of MENTHOR identify compounds for which modeling is needed. It also forms the data to be searched with ALLADDIN, our substructure/geometric search program. MENTHOR expedites molecular modeling by organizing previous work and facilitating transmission of information between individuals. Examples from modeling of D-2 receptor agonists are shown.

The metabolism of tritiated benzo[a]pyrene (B[a]P) by primary mouse embryo cells in culture was studied. At concentrations of B[a]P in the medium below about 2–3 mμ moles/ml, metabolism was exponential with time, but at higher concentrations a period of rapid metabolism was followed by a progressive decline. The extent of binding of the hydrocarbon to DNA, RNA and protein was studied under the above conditions. It was found that at low doses of hydrocarbon the binding to macromolecules was proportional to the overall metabolism, with the result that the binding index, i.e. the amount of hydrocarbon bound divided by the amount metabolized, was constant. At higher dose levels, overall metabolism did increase with dose whereas macromolecular binding reached a plateau value. This resulted in a fall in the “binding index” below the constant value found at low doses of B[a]P. As a consequence of these results and those of other workers, a model scheme is proposed for the enzymic metabolism of B[a]P which accords with the known facts of metabolism and macromolecular binding. RELATION DU METABOLISME ET DE LA LIAISON MACROMOLECULAIRE DU CARCINOEGNE BENZO[A]PYReNE, D'APRES DES CULTURES DE CELLULES EMBRYONNAIRES DE SOURIS Nous avons etudie le metabolisme du benzo[a]pyrkne [a] (B [a] P ) tritie dans des cultures primitives de cellules d'embryon de souris. Lorsque la concentration de B [a] P dans le milieu est infe'rieure a environ 2 ou 3 mp moles/ml, le me'tabolisme est exponentiel avec le temps mais, 2 de plus fortes concentrations, une pdriode de metabolisme rapide est suivie d'un de'clin progressif. Nous avons Ctudie' l'importance de la liaison de l'hydrocarbure a l'ADN, a I'ARN et aux protdines dans les conditions susmentionne'es. Nous avons constate' qu'a de faibles doses d'hydrocarbure la liaison avec les macromole'culm est proportionnelle au me'tabolisme global, ce qui fait que I'indice de liaison, c'est-a-dire la quantitk d'hydrocarbure lie' divisde par la quantitk me'tabolise'e, ne varie pas. A de plus fortes doses, le mttabolisme global augmente en měme temps que la dose alors que la liaison macromoleculaire atteint un palier, ce qui entraǐne une chute de “l'indice de liaison” qui est alors inferieur a la valeur constante observee avec de faibles doses de B [a] P. Au vu de ces resultats et des constatations d'autres chercheurs, nous proposons, pour le metabolisme enzymatique du B [a] P, un modele de tableau qui concorde avec les aspects connus du metabolisme et de la liaison macromoleculaire.

Human desialylated transcortin was found to be microheterogeneous by isoelectric focusing. All samples tested showed two cortisol-binding components. Liganded with cortisol these components are isoelectric at pH 5.2 and 5.4. In the unliganded form, however, they eluted at pH 5.4 and 5.6, respectively. This cortisol-controlled transition was reversible in both directions. Isolated components containing trace amounts of [3H]cortisol accumulate the steroid in the position of the corresponding liganded form at the anodic side of the unliganded binder. Electrofocusing of desialylated plasma containing [3H]cortisol results in a cortisol accumulation at pH 5.1 at the anodic side of the unliganded binding components. The experiments show that electrofocusing can profitably be used to study ligand-protein interactions and that radioligand detection is not always an adequate method to localize a macromolecular binding agent.

The state of calcium and inorganic orthophosphate was investigated in resting parotid saliva and in stimulated parotid, submaxillary and whole saliva. Macromolecular binding was determined utilizing ultrafiltration procedures. Ionized calcium and phosphate in the ultrafiltrates were calculated on the basis of chemical analyses and considering the formation of the ion pairs CaHPO 4 o , CaH 2 PO 4 + and CaHCO 3 + . Mean findings showed that 85 per cent of the calcium in resting parotid saliva was ionized, while 8 per cent was bound to macromolecules and 6 per cent in ion pairs. In stimulated secretions, the ionized calcium ranged from 45 per cent (submaxillary saliva) to 54 per cent (mixed whole saliva) of total calcium. The macromolecular bound fraction accounted for 30–43 per cent while ion pairs bound 12–17 per cent of the calcium. Mean concentrations of ionized calcium were 0.74, 0.49, 0.76 and 0.59 mM respectively for the four types of saliva. Ultrafiltrability of inorganic orthophosphate ranged from 66 to 70 per cent in stimulated salivas and was over 90 per cent in resting parotid saliva. The ultrafiltrable phosphate was almost fully ionized because only 1–2 per cent was complexed with calcium. Mean concentrations of ionized orthophosphate were 6.58,2.59,2.08 and 2.53 mM respectively for the four types of saliva.

The use of molecular simulation to estimate the strength of macromolecular binding free energies is becoming increasingly widespread, with goals ranging from lead optimization and enrichment in drug discovery to personalizing or stratifying treatment regimes. In order to realize the potential of such approaches to predict new results, not merely to explain previous experimental findings, it is necessary that the methods used are reliable and accurate, and that their limitations are thoroughly understood. However, the computational cost of atomistic simulation techniques such as molecular dynamics (MD) has meant that until recently little work has focused on validating and verifying the available free energy methodologies, with the consequence that many of the results published in the literature are not reproducible. Here, we present a detailed analysis of two of the most popular approximate methods for calculating binding free energies from molecular simulations, molecular mechanics Poisson–Boltzmann surf...

The dissociation of ligands from proteins and other biomacromolecules occurs over a wide range of timescales. For most pharmaceutically relevant inhibitors, these timescales are far beyond those that are accessible by conventional molecular dynamics (MD) simulation. Consequently, to explore ligand egress mechanisms and compute dissociation rates, it is necessary to enhance the sampling of ligand unbinding. Random Acceleration MD (RAMD) is a simple method to enhance ligand egress from a macromolecular binding site, which enables the exploration of ligand egress routes without prior knowledge of the reaction coordinates. Furthermore, the tauRAMD procedure can be used to compute the relative residence times of ligands. When combined with a machine-learning analysis of protein-ligand interaction fingerprints (IFPs), molecular features that affect ligand unbinding kinetics can be identified. Here, we describe the implementation of RAMD in GROMACS 2020, which provides significantly improved computational performance, with scaling to large molecular systems. For the automated analysis of RAMD results, we developed MD-IFP, a set of tools for the generation of IFPs along unbinding trajectories and for their use in the exploration of ligand dynamics. We demonstrate that the analysis of ligand dissociation trajectories by mapping them onto the IFP space enables the characterization of ligand dissociation routes and metastable states. The combined implementation of RAMD and MD-IFP provides a computationally efficient and freely available workflow that can be applied to hundreds of compounds in a reasonable computational time and will facilitate the use of tauRAMD in drug design., Comment: 35 pages, 5 figures in the main text and 9 figures in the supplementary information. The following article has been submitted to The Journal of Chemical Physics. After it is published, it will be found at https://publishing.aip.org/resources/librarians/products/journals/