Modulation of the enzymatic activity of papain by interdomain residues remote from the active site

The two main catalytic residues Cys25 and Hisl59 of the monomericcysteine protease papain are located on different walls of acleft formed by two domains. This topology suggests a possiblerelationship between relative domain organization and catalyticmechanism. The effect on enzymatic parameters of structuralmodifications at various locations of the twodomain interfaceof papain was examined by individual or double replacementsby Ala of pairs of interacting residues. Most modificationshad no effect on enzyme activity. However, the enzyme's substrateturnover (k_cat) decreased following simultaneous alterationof the two most conserved residues, forming an apolar contactlocated 15 Å away from the active site. The pH activityprofile of the double mutant was unchanged, indicating a conservedionization state of the active site thiolate-imidazolium ionpair. This state is strongly dependent on the distance separatingthe two residues, thus suggesting that the active site geometryhas not been significantly altered. Efficient enzymatic activityin papain requires more than a correct active site geometryand is influenced by domain packing properties in a region remotefrom the active site.     

Thioether Analogues of Disulfide‐Bridged Cyclic Peptides Targeting Death Receptor 5: Conformational Analysis,Dimerisation and Consequences for Receptor Activation

Cyclic peptides containing redox‐stable thioether bridges might provide a useful alternative to disulfide‐bridged bioactive peptides. We report the effect of replacing the disulfide bridge with a lanthionine linkage in a 16‐mer cyclic peptide that binds to death receptor 5 (DR5, TRAIL‐R2). Upon covalent oligomerisation, the disulfide‐bridged peptide has previously shown similar behaviour to that of TNF‐related apoptosis inducing ligand (TRAIL), by selectively triggering the DR5 cell death pathway. The structural and biological properties of the DR5‐binding peptide and its desulfurised analogue were compared. Surface plasmon resonance (SPR) data suggest that these peptides bind DR5 with comparable affinities. The same holds true for dimeric versions of these peptides: the thioether is able to induce DR5‐mediated apoptosis of BJAB lymphoma and tumorigenic BJELR cells, albeit to a slightly lower extent compared to its disulfide homologue. NMR analysis revealed subtle variation in the conformations of the two peptides and suggests that the thioether peptide is slightly less folded than its disulfide homologue. These observations could account for the different capability of the two dimers to cluster DR5 receptors on the cell surface and to trigger apoptosis. Nevertheless, our results suggest that the thioether peptide is a potential candidate for evaluation in animal models.     

Coordinated amino acid changes in homologous protein families

In the tobamovirus coat protein family, amino acid residuesat some spatially close positions are found to be substitutedin a coordinated manner [Altschuh et al. (1987) J. Mol. Biol.,193,693]. Therefore, these positions show an identical patternof amino acid substitutions when amino acid sequences of thesehomologous proteins are aligned. Based on this principle, coordinatedsubstitutions have been searched for in three additional proteinfamilies: serine proteases, cysteine proteases and the haemoglobins.Coordinated changes have been found in all three protein familiesmostly within structurally constrained regions. This methodworks with a varying degree of success depending on the functionof the proteins, the range of sequence similarities and thenumber of sequences considered. By relaxing the criteria forresidue selection, the method was adapted to cover a broaderrange of protein families and to study regions of the proteinshaving weaker structural constraints. The information derivedby these methods provides a general guide for engineering ofa large variety of proteins to analyse structure–functionrelationships.     

Small-molecule negative modulators of adrenomedullin: design, synthesis, and 3D-QSAR study

Adrenomedullin (AM) is a peptidic hormone that was isolated in 1993, the function of which is related to several diseases such as diabetes, hypertension, and cancer. Compound 1 is one of the first nonpeptidic small-molecule negative modulators of AM, identified in a high-throughput screen carried out at the National Cancer Institute. Herein we report the synthesis of a series of analogues of 1. The ability of the synthesized compounds to disrupt the binding between AM and its monoclonal antibody has been measured, together with surface plasmon resonance (SPR)-based binding assays as implemented with Biacore technology. These data were used to derive a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, with a q(2) (LOO) value of 0.8240. This study has allowed us to identify relevant features for effective binding to AM: the presence of a hydrogen-bond donor group and an aromatic ring. Evaluation of the ability of selected compounds to modify cAMP production in Rat2 cells showed that the presence of a free carboxylic acid is essential for negative AM modulation.     

QSAR studies applied to the prediction of antigen-antibody interaction kinetics as measured by BIACORE

The objective of this work was to investigate the potentialof the quantitative structure–activity relationships (QSAR)approach for predictive modulation of molecular interactionkinetics. A multivariate QSAR approach involving modificationsin peptide sequence and buffer composition was recently usedin an attempt to predict the kinetics of peptide–antibodyinteractions as measured by BIACORE. Quantitative buffer–kineticsrelationships (QBKR) and quantitative sequence–kineticsrelationships (QSKR) models were developed. Their predictivecapacity was investigated in this study by comparing predictedand observed kinetic dissociation parameters (k_d) for new antigenicpeptides, or in new buffers. The range of experimentally measuredk_d variations was small (300-fold), limiting the practical valueof the approach for this particular interaction. However, themodels were validated from a statistical point of view. In QSKR,the leave-one-out cross validation gave Q² = 0.71 for 24 peptides(all but one outlier), compared to 0.81 for 17 training peptides.A more precise model (Q² = 0.92) could be developed when removingsets of peptides sharing distinctive structural features, suggestingthat different peptides use slightly different binding modes.All models share the most important factor and are informativefor structure–kinetics relationships. In QBKR, the measuredeffect on k_d of individual additives in the buffers was consistentwith the effect predicted from multivariate buffers. Our resultsopen new perspectives for the predictive optimization of interactionkinetics, with important implications in pharmacology and biotechnology.     

Progress Report on Phase Separation in Polymer Solutions

Polymeric porous media (PPM) are widely used as advanced materials, such as sound dampening foams, lithium‐ion batteries, stretchable sensors, and biofilters. The functionality, reliability, and durability of these materials have a strong dependence on the microstructural patterns of PPM. One underlying mechanism for the formation of porosity in PPM is phase separation, which engenders polymer‐rich and polymer‐poor (pore) phases. Herein, the phase separation in polymer solutions is discussed from two different aspects: diffusion and hydrodynamic effects. For phase separation governed by diffusion, two novel morphological transitions are reviewed: “cluster‐to‐percolation” and “percolation‐to‐droplets,” which are attributed to an effect that the polymer‐rich and the solvent‐rich phases reach the equilibrium states asynchronously. In the case dictated by hydrodynamics, a deterministic nature for the microstructural evolution during phase separation is scrutinized. The deterministic nature is caused by an interfacial‐tension‐gradient (solutal Marangoni force), which can lead to directional movement of droplets as well as hydrodynamic instabilities during phase separation.     

Estimates in deterministic fate modelling of environmental chemicals

Ecological modelling is frequently confronted with the problem of unavailable parameters, or input data of poor quality. This makes the interpretation of the results difficult, and predictions questionable, unless the quality of the parameters is taken into account. In this paper, we propose a mathematical approach to address the problem of large parameter variability in deterministic models, and especially in linear compartmental models. The method is based on a classical comparison theorem for differential equations, and yields upper and lower estimates for solutions when upper and lower estimates for the model input functions are given. Such estimates may be checked against experimental data, and thus play a crucial role in verification or falsification of model predictions. Applications are presented with regard to fate modelling of environmental chemicals in a pond sediment.     

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates

Predictive engineering of antibodies exhibiting fast kineticproperties could provide reagents for biotechnological applicationssuch as continuous monitoring of compounds or affinity chromatography.Based on covariance analysis of murine germline antibody variabledomains, we selected position L34 (Kabat numbering) for mutationalstudies. This position is located at the VL/VH interface, atthe base of the paratope but with limited antigen contacts,thus making it an attractive position for mild alterations ofantigen binding properties. We introduced a serine at positionL34 in two different antibodies: Fab (fragment antigen binding)57P (Asn34Ser) and scFv (single chain fragment variable) 1F4(Gln34Ser), that recognize peptides derived from the coat proteinof tobacco mosaic virus and the oncoprotein E6, respectively.Both mutated antibodies exhibited similar properties: (i) expressionlevels of active fragments in Escherichia coli were markedlyimproved; (ii) thermostability was enhanced; and (iii) dissociationrate parameters (k_off) were increased by 2- and at least 57-foldfor scFv1F4 and Fab57P, respectively, while their associationrate parameters (k_on) remained unchanged. The L34 Ala and Thrmutants of both antibody fragments did not possess these properties.This first demontration of similar effects observed in two antibodieswith different specificities may open the way to the predictivedesign of molecules with enhanced stability and fast dissociationrates. Received October 3, 2002; revised March 7, 2003; accepted April 4, 2003.