Physicochemical Constraints Research Articles

A Bird's-Eye View of Enzyme Evolution: Chemical, Physicochemical, and Physiological Considerations.

Enzymes catalyze a vast range of reactions. Their catalytic performances, mechanisms, global folds, and active-site architectures are also highly diverse, suggesting that enzymes are shaped by an entire range of physiological demands and evolutionary constraints, as well as by chemical and physicochemical constraints. We have attempted to identify signatures of these shaping demands and constraints. To this end, we describe a bird's-eye view of the enzyme space from two angles: evolution and chemistry. We examine various chemical reaction parameters that may have shaped the catalytic performances and active-site architectures of enzymes. We test and weigh these considerations against physiological and evolutionary factors. Although the catalytic properties of the "average" enzyme correlate with cellular metabolic demands and enzyme expression levels, at the level of individual enzymes, a multitude of physiological demands and constraints, combined with the coincidental nature of evolutionary processes, result in a complex picture. Indeed, neither reaction type (a chemical constraint) nor evolutionary origin alone can explain enzyme rates. Nevertheless, chemical constraints are apparent in the convergence of active-site architectures in independently evolved enzymes, although significant variations within an architecture are common.

Dimethyl ether to hydrocarbons over ZSM-5: Kinetic study in an external recycle reactor

DME provides advantages in the valorization of feed streams of low H2/CO ratio, which are formed from feedstocks such as biomass or coal.It is currently accepted that the conversion of oxygenates, such as DME or methanol, to hydrocarbons proceeds via the dual-cycle mechanism.After inspection of several kinetic models developed for the methanol-to-gasoline (MtG) reaction, we show in this investigation that they have limited applicability when dimethyl ether (DME) is used as feedstock. Therefore, the development of a kinetic model able to represent the DME-to-hydrocarbons (DtH) over a ZSM-5 catalyst was necessary.In this research, we have developed a new kinetic model that considers main steps of the dual cycle mechanism, including: (i) formation of aromatic intermediates (polymethylbenzenes); (ii) dealkylation of the intermediates to produce ethylene and propylene; (iii) methylation of small olefins to increase the chain size of the hydrocarbons; (iv) hydrogenation of ethylene, propylene and butenes to produce the corresponding saturated compounds; and (v) dimerization reactions between propylene and butenes to produce higher hydrocarbons. Moreover, the kinetic parameters of this new model were estimated from regression analysis using kinetic data measured under gradientless conditions in a fixed-bed external recycle reactor over a wide range of conditions, with temperatures from 325 to 375 °C, weight hourly space velocities (WHSV) from 25 to 125 h−1, a total pressure of 1 bar and undiluted DME as feed. The newly developed model allowed a good description of the experimental results, showed better performance than models available in literature for the MtG reaction, and rendered kinetic parameters that met physicochemical and statistical constraints, showing good agreement with results from DFT calculations reported by other authors.

PyTFA and matTFA: a Python package and a Matlab toolbox for Thermodynamics-based Flux Analysis.

SummarypyTFA and matTFA are the first published implementations of the original TFA paper. Specifically, they include explicit formulation of Gibbs energies and metabolite concentrations, which enables straightforward integration of metabolite concentration measurements.MotivationHigh-throughput analytic technologies provide a wealth of omics data that can be used to perform thorough analyses for a multitude of studies in the areas of Systems Biology and Biotechnology. Nevertheless, most studies are still limited to constraint-based Flux Balance Analyses (FBA), neglecting an important physicochemical constraint: thermodynamics. Thermodynamics-based Flux Analysis (TFA) in metabolic models enables the integration of quantitative metabolomics data to study their effects on the net-flux directionality of reactions in the network. In addition, it allows us to estimate how far each reaction operates from thermodynamic equilibrium, which provides critical information for guiding metabolic engineering decisions.ResultsWe present a Python package (pyTFA) and a Matlab toolbox (matTFA) that implement TFA. We show an example of application on both a reduced and a genome-scale model of E. coli., and demonstrate TFA and data integration through TFA reduce the feasible flux space with respect to FBA.Availability and implementationDocumented implementation of TFA framework both in Python (pyTFA) and Matlab (matTFA) are available on www.github.com/EPFL-LCSB/.Supplementary information Supplementary data are available at Bioinformatics online.

Physicochemical Drivers of Microbial Community Structure in Sediments of Lake Hazen, Nunavut, Canada

The Arctic is undergoing rapid environmental change, potentially affecting the physicochemical constraints of microbial communities that play a large role in both carbon and nutrient cycling in lacustrine environments. However, the microbial communities in such Arctic environments have seldom been studied, and the drivers of their composition are poorly characterized. To address these gaps, we surveyed the biologically active surface sediments in Lake Hazen, the largest lake by volume north of the Arctic Circle, and a small lake and shoreline pond in its watershed. High-throughput amplicon sequencing of the 16S rRNA gene uncovered a community dominated by Proteobacteria, Bacteroidetes, and Chloroflexi, similar to those found in other cold and oligotrophic lake sediments. We also show that the microbial community structure in this Arctic polar desert is shaped by pH and redox gradients. This study lays the groundwork for predicting how sediment microbial communities in the Arctic could respond as climate change proceeds to alter their physicochemical constraints.

Hydrothermal fluid evolution in the ‘Botro ai Marmi’ quartz-monzonitic intrusion, Campiglia Marittima, Tuscany, Italy. Evidence from a fluid-inclusion investigation

ABSTRACTThe quartz-monzonitic intrusion of ‘Botro ai Marmi’ in Tuscany, Italy, can be considered to be a typical example of an intrusion-centred magmatic hydrothermal system. The evolution of hydrothermal fluids in the ‘Botro ai Marmi’ intrusion was investigated using fluid-inclusion analyses to provide suitable physico-chemical constraints on the fluids involved in the late- to post-magmatic hydrothermal activity that affected the intrusion, providing inferences on their origin and variations of temperature and pressure with time.This work demonstrates that the earliest fluids circulating in the ‘Botro ai Marmi’ intrusion were high-temperature brines exsolved directly from the crystallizing magma. This fluid circulated in the intrusion under lithostatic conditions (P > 90 MPa, T > 540°C). A second evolutionary stage of the magmatic hydrothermal system is marked by the transition from lithostatic (>90 MPa) to hydrostatic dominated conditions (50 to 10 MPa). In this stage the fluids are also interpreted to be mainly orthomagmatic in origin but unmixed in a high-salinity brine and in a low-salinity vapour aqueous phase, at a temperature ranging from ~500 to 300°C. These fluids were responsible for the potassic alteration facies. At a later stage of hydrothermal evolution, abundant meteoric dominated fluids entered the system and are associated with propylitic alteration. This event marks the transition from a magmatic-hydrothermal system to a typical hydrothermal (‘geothermal’) system, which can be assumed to be similar to some extent to the nearby active high-enthalpy geothermal system of Larderello. Low-temperature and low-salinity meteoric water-dominated fluids characterize the latest stage of the ‘Botro ai Marmi’ hydrothermal system.

Mineralogy of Bi-sulfosalts and tellurides from the Yaoan gold deposit, southwest China: Metallogenic implications

Bismuth-sulfosalts and -tellurides are commonly present in many hydrothermal gold deposits and can provide important genetic and physicochemical constraints on the metallogenesis. The abundant auriferous specular hematite veins in the Yaoan gold deposit of the Jinshajiang-Red River alkaline porphyry metallogenic belt (southwest China) contain an assemblage of Bi-sulfosalts and tellurides together with hematite, pyrite, and chalcopyrite. Bismuth-sulfosalts are characterized by replacement textures resulting from changes of fluid composition (e.g., Pb, Bi, Cu, and Ag) and mainly include bismuthinite derivatives (e.g., bismuthinite, paarite, krupkaite, lindströmite, and friedrichite-aikinite), paděraite, cuprobismutite homologues (probably submicroscopic intergrowths of hodrushite and cuprobismutite), and emplectite. Tellurides are tiny, comprising tsumoite, melonite, hessite, and calaverite, which coexist with bismuthinite derivatives. These Bi-sulfosalts and tellurides have direct contact with gold, indicating that the Yaoan gold metallogenesis was likely linked to the syenite porphyry magmatism that was originally sourced from a regional lithospheric mantle probably metasomatized by subduction of Te-rich sediments. The telluride assemblage also reveals that the gold was precipitated under oxidizing conditions that has a high tellurium fugacity at the beginning (fTe2 = ∼10−7.9 and fS2 = ∼10−8.5 at ∼300 °C).

Methanol to dimethyl ether conversion over a ZSM-5 catalyst: Intrinsic kinetic study on an external recycle reactor

In this work, several kinetic models have been studied for the methanol dehydration to dimethyl ether (DME), applying a commercial ZSM-5 catalyst rather than state-of-the-art alumina.The kinetic tests were carried out in a fixed-bed external-recycle reactor, in the absence of temperature and concentration gradients.Kinetic parameters of the different models were calculated and evaluated against physicochemical constraints and guidelines on entropy values. After discrimination, it was concluded that a modification of a model previously developed by Klusáček & Schneider for methanol dehydration over different catalyst formulations than alumina or zeolites describes best the methanol dehydration reaction, with excellent agreement between experimental data and calculated values. The selection of this model supports a mechanism in which methanol experiences dissociative adsorption and a surface reaction is the rate determining step (RDS) of the catalytic cycle.The kinetic measurements were performed at a total pressure of 1 bar, partial pressure of methanol (pm) from 0.3 bar to 1 bar, temperatures from 140 °C to 190 °C, weight hourly space velocities (WHSV) from 15 h−1 to 100 h−1, and two different amounts of water content (0 and 30 wt%). Experiments at different methanol concentrations in the feed revealed a weak dependence of the rate on pm. It could be determined to our knowledge for the first time that the reaction order with respect to water is negative, in the range of −0.80 to 0, thus providing quantitative proof of the inhibition effect of water on the methanol dehydration reaction rate over ZSM-5 catalysts, while DME showed no effect.

Understanding the impact of soil sodicity on mycorrhizal symbiosis: Some facts and gaps identified from cotton systems

Soil sodicity degrades land and more than half of the world’s sodic soils are in Australia. Farmers in Australia produce and export cotton grown in sodic soils. Undesirable physicochemical constraints (e.g. high pH, high bulk density, low porosity and reduced oxygen content) are associated with sodic soils and may adversely affect microbial interactions in the rhizosphere, including mycorrhizal associations. This viewpoint focusses on facts and gaps in our knowledge about mycorrhizal associations in sodic soils, with special attention to cotton systems. We highlight the difference between saline and sodic soil conditions and the impacts they may have on host plant-mycorrhizae symbiosis. This viewpoint identifies the need for more research on the potential impact of sodicity on mycorrhizal species diversity, functionality and benefits to crop growth. Changes in agronomic management strategies to maximize mycorrhizal symbiosis benefits are suggested, especially for those plant species, like cotton, that are highly reliant on mycorrhizal symbiosis for optimal growth and nutrient uptake.

Repeated Stand-Replacing Crown Fires Affect Seed Morphology and Germination in Aleppo pine.

Post-fire reproductive niche of Aleppo pine (Pinus halepensis) is deeply interlaced with fire products. Indeed, the high pH and low osmotic potentials of ash beds under burnt crowns constitute the main constraints to seed germination. In this study, we aim to investigate whether fire recurrence, through the physico-chemical constraints imposed by the ash beds, affects the reproduction ability of P. halepensis at the germination stage. To this aim, Aleppo pine seeds were collected in neighboring even-aged stands subjected to 0, 1, or 2 fires (namely fire cohorts), and seed morphology and germination performance, in terms of cumulative germination and germination kinetics, were studied under increasing osmotic potentials (from 0.0 to −1.2 MPa) and pH (from 6 to 11). Besides fire history, the role of ontogenetic age of mother plants on seed morphology and germination was also investigated. Differences in seed morphology among the three cohorts have been highlighted in a multivariate context, with anisotropic enlargement of the seeds produced by pine stands experiencing repeated fires. The patterns of seed germination varied primarily in relation to the fire cohort, with seeds from the pine stand experiencing repeated fires exhibiting enhanced tolerance to pH stress. Conversely, germination performances under osmotic constraints mainly depends on tree ontogenetic stage, with an involvement of fire history especially in the timing of seed germination. Our results suggest that, at least in the short term, fire recurrence does not constrain the reproduction ability of Aleppo pine. These results highlight the need for further research to elucidate the mechanisms behind these responses to recurrent fires.

Molecular Dynamics as a tool for in silico screening of skin permeability

Prediction of skin permeability can have manifold applications ranging from drug delivery to toxicity prediction. Along with the semi-empirical or mechanistic models proposed in the last decades, Molecular Dynamics simulations have recently become a fruitful tool for investigating membrane permeability, in particular as they allow the involved mechanisms to be modelled at a molecular level. Despite their significant structural complexity, Molecular Dynamics simulations can also be utilized to study permeation through the lipid matrix that characterizes the stratum corneum. In this work, Steered Molecular Dynamics simulations are performed on a suitably developed stratum corneum lipid matrix model. Regardless of their actual tortuous path within the stratum corneum, the permeants, taken from a Fully Validated dataset of 80 compounds of known permeability coefficient, are moved through the bilayer along its normal. This allows the exploration of all the possible conformational and physicochemical constraints the molecule experiences when moving through the bilayer. The so performed Steered Molecular Dynamics simulations are then utilized to extract the corresponding lipophilicity and diffusion parameters as computed by subdividing the entire path in 18 regions of different polarity and composition. Correlative analyses showed that the water-lipids interface is the best performing region and that significant enhancements can be gained by including parameters accounting for the temperature effect. Taken together, the developed models possess an enhanced predictive power compared to the existing equations and statistics are approaching the best possible results, given the uncertainty in the utilized permeability data.

Physicochemical and Nutritional Requirements for Axenic Replication Suggest Physiological Basis for Coxiella burnetii Niche Restriction.

Bacterial obligate intracellular parasites are clinically significant animal and human pathogens. Central to the biology of these organisms is their level of adaptation to intracellular replication niches associated with physicochemical and nutritional constraints. While most bacterial pathogens can adapt to a wide range of environments, severe niche restriction—an inability to thrive in diverse environments—is a hallmark of bacterial obligate intracellular parasites. Herein the physicochemical and nutritional factors underlying the physiological basis for niche restriction in the zoonotic bacterial obligate intracellular parasite and Q fever agent Coxiella burnetii are characterized. Additionally, these factors are reviewed in the context of C. burnetii evolution and continued (patho) adaptation. C. burnetii replication was strictly dependent on a combination of moderately acidic pH, reduced oxygen tension, and presence of carbon dioxide. Of macronutrients, amino acids alone support replication under physicochemically favorable conditions. In addition to utilizing gluconeogenic substrates for replication, C. burnetii can also utilize glucose to generate biomass. A mutant with a disruption in the gene pckA, encoding phosphoenolpyruvate carboxykinase (PEPCK), the first committed step in gluconeogenesis, could be complemented chemically by the addition of glucose. Disruption of pckA resulted in a moderate glucose-dependent growth defect during infection of cultured host cells. Although, C. burnetii has the theoretical capacity to synthesize essential core metabolites via glycolysis and gluconeogenesis, amino acid auxotrophy essentially restricts C. burnetii replication to a niche providing ample access to amino acids. Overall, the described combination of physiochemical and nutritional growth requirements are strong indicators for why C. burnetii favors an acidified phagolysosome-derived vacuole in respiring tissue for replication.

How Cells Can Control Their Size by Pumping Ions.

The ability of all cells to set and regulate their size is a fundamental aspect of cellular physiology. It has been known for sometime but not widely so, that size stability in animal cells is dependent upon the operation of the sodium pump, through the so-called pump-leak mechanism (Tosteson and Hoffman, 1960). Impermeant molecules in cells establish an unstable osmotic condition, the Donnan effect, which is counteracted by the operation of the sodium pump, creating an asymmetry in the distribution of Na+ and K+ staving off water inundation. In this paper, which is in part a tutorial, I show how to model quantitatively the ion and water fluxes in a cell that determine the cell volume and membrane potential. The movement of water and ions is constrained by both osmotic and charge balance, and is driven by ion and voltage gradients and active ion transport. Transforming these constraints and forces into a set of coupled differential equations allows us to model how the ion distributions, volume and voltage change with time. I introduce an analytical solution to these equations that clarifies the influence of ion conductances, pump rates and water permeability in this multidimensional system. I show that the number of impermeant ions (x) and their average charge have a powerful influence on the distribution of ions and voltage in a cell. Moreover, I demonstrate that in a cell where the operation of active ion transport eliminates an osmotic gradient, the size of the cell is directly proportional to x. In addition, I use graphics to reveal how the physico-chemical constraints and chemical forces interact with one another in apportioning ions inside the cell. The form of model used here is applicable to all membrane systems, including mitochondria and bacteria, and I show how pumps other than the sodium pump can be used to stabilize cells. Cell biologists may think of electrophysiology as the exclusive domain of neuroscience, however the electrical effects of ion fluxes need to become an intimate part of cell biology if we are to understand a fundamental process like cell size regulation.

Physicochemical Constraints on Surfactant Blends under Harsh Conditions and Evaluation of a Proposed Solution

Chemical-enhanced oil recovery has been applied successfully in reservoirs with mild salinity and temperature conditions. Offshore reservoirs challenge chemical flooding, e.g., low-tension and foam flooding, because of the combined hardness and salinity of seawater along with the characteristics of the reservoir connate brine. These physicochemical conditions impose severe limitations on adequate phase behavior for most commercial surfactants. The purpose of this research is to analyze surfactant phase behavior for scenarios with seawater as the main carrying fluid for surfactant systems for harsh conditions similar to those in heavy-oil reservoirs in the Gulf of Mexico. We also aim at testing a recently developed surfactant characterization technique using a recently published NMR protocol [Garcia-Olvera et al., Energy Fuels, 2016, 30, 63]. In this technique, NMR spectra of surfactants, cosurfactants, and polymers are calibrated to enable analysis of individual components in chemical blends. Critical mic...

Weed Suppression and Performance of Grain Legumes Following an Irrigated Rice Crop in Southern Australia

Post-rice irrigated soils offer several potential advantages for the growth of subsequent crops, but Australian producers have often been reluctant to grow grain legumes immediately following a rice crop due to physico-chemical constraints. A field experiment was thus conducted to explore the potential for producing grain legumes following rice in comparison to those following a fallow during 2012 and 2013. Two grain legumes, field pea and faba bean, were sown 5, 7 and 12 weeks after rice harvest in 2013 at Yanco, NSW, and plant growth indicators and grain yield were compared. Early sowing of field pea following rice gave the best outcome, with plants flowering three weeks earlier and yielding 1330 kg·ha−1 more grain than after fallow. In contrast, faba bean yield was 35 kg·ha−1 less after rice than after fallow across the three sowing dates. Higher pea yield was consistent with the early emergence of seedlings, higher light interception and overall greater plant growth following rice. Post-rice crops also had 10-fold less weed infestation than crops in a similarly-established fallow treatment and, thus, required far less weed management. Legume crops sown at the later seeding date had significantly reduced (~50%–60%) yields compared to those of the first two sowings; this is most likely a reflection of reduced temperatures and day lengths experienced during vegetative and reproductive growth phases.

A Pareto Algorithm for Efficient De Novo Design of Multi-functional Molecules.

We have introduced a Pareto sorting algorithm into Synopsis, a de novo design program that generates synthesizable molecules with desirable properties. We give a detailed description of the algorithm and illustrate its working in 2 different de novo design settings: the design of putative dual and selective FGFR and VEGFR inhibitors, and the successful design of organic structure determining agents (OSDAs) for the synthesis of zeolites. We show that the introduction of Pareto sorting not only enables the simultaneous optimization of multiple properties but also greatly improves the performance of the algorithm to generate molecules with hard-to-meet constraints. This in turn allows us to suggest approaches to address the problem of false positive hits in de novo structure based drug design by introducing structural and physicochemical constraints in the designed molecules, and by forcing essential interactions between these molecules and their target receptor.

Small size of membrane-tethered ligands regulates kinase-phosphatase segregation in γδ TCR triggering.

Abstract Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not undergo canonical thymic selection, and consequently are not restricted by pMHC antigens or the co-receptors CD4/8. Some of these unconventional T cells recognize invariant membrane-tethered ligands, and their rapid activation and recruitment to sites of inflammation serves as a ‘first line of defense’ against incipient infection. The substantial structural and stoichiometric similarities between αβ and γδ TCRs prompted us to investigate whether γδ TCR ligand-recognition and triggering shared basic physicochemical constraints with conventional αβ TCRs. We chose as a model system the stress-inducible murine surface protein T22 that is recognized by the G8 γδ TCR - for which detailed biophysical and structural information is available. Since T22 is similar in size to pMHC molecules, we tested whether ‘size-based’ kinase-phosphatase segregation, following G8 engagement by T22, controls initiation of TCR signaling. In our ongoing studies, we have established that the small axial size of T22 is critical for G8 triggering. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 responses. Using optical and electron microscopy we show that elongated T22/G8 complexes increases access of inhibitory phosphatases to sites of TCR engagement at cell-cell contacts. We are testing whether truncation of the ectodomain of the large cell surface phosphatases CD45/CD148, which are normally sterically segregated from engaged TCR, affects triggering. Finally, we are adapting these strategies, to investigate mechanisms of triggering by γδ T cells that recognize small-molecule organic compounds.

Smart ore blending methodology for ferromanganese production process

This study is carried out to develop a smart ore blending methodology for high carbon ferromanganese production units. Geometallurgical characterisation of the ores collected from 10 different mines has been carried out to estimate variation in their aptness for the alloy production process. An evolutionary algorithm-based methodology has been adopted for ore blending to maximise the total manganese content of the ore blend under applied physico-chemical constraints. Analysis provides various blends of the same chemical composition but of different geometallurgical ranks. An artificial neural network model has been developed to predict the operational parameters such as slag and metal composition. This method allows the operator to visualise the impact of combinations of different ore blends on slag–metal composition as well as on electric power and coke required for smelting reduction of ores in the submerged arc furnace. It was observed that best blends could reduce power and coke consumption by 100 kWh ton−1 and 30 kg ton−1, respectively, but optimum values can be established in long run considering conservation of natural resources.

3D numerical modeling of mantle flow, crustal dynamics and magma genesis associated with slab roll-back and tearing: The eastern Mediterranean case

Interactions between subduction dynamics and magma genesis have been intensely investigated, resulting in several conceptual models derived from geological, geochemical and geophysical data. To provide physico-chemical constraints on these conceptual models, self-consistent numerical simulations containing testable thermo-mechanical parameters are required, especially considering the three-dimensional (3D) natural complexity of subduction systems. Here, we use a 3D high-resolution petrological and thermo-mechanical numerical model to quantify the relative contribution of oceanic and continental subduction/collision, slab roll-back and tearing to magma genesis and transport processes. Our modeling results suggest that the space and time distribution and composition of magmas in the overriding plate is controlled by the 3D slab dynamics and related asthenospheric flow. Moreover, the decrease of the bulk lithospheric strength induced by mantle- and crust-derived magmas promotes the propagation of strike-slip and extensional fault zones through the overriding crust as response to slab roll-back and continental collision. Reduction of the lithosphere/asthenosphere rheological contrast by lithospheric weakening also favors the transmission of velocities from the flowing mantle to the crust. Similarities between our modeling results and the late Cenozoic tectonic and magmatic evolution across the eastern Mediterranean region suggest an efficient control of mantle flow on the magmatic activity in this region, which in turn promotes lithospheric deformation by mantle drag via melt-induced weakening effects.

Therapeutic actions of curcumin in bone disorders.

Curcumin is the active component of turmeric extract derived from the Curcuma longa plant. In the last decade, curcumin has raised a considerable interest in medicine owing to its negligible toxicity and multiple therapeutic actions including anti-cancer, anti-inflammatory and anti-microbial activities. Among the various molecular targets of curcumin, some are involved in bone remodeling, which strongly suggests that curcumin can affect the skeletal system. The review sheds light on the current and potential applications of curcumin to treat bone disorders characterized by an excessive resorption activity. Within the scope of this review, the novel formulations of curcumin to overcome its physico-chemical and pharmacokinetic constraints are also discussed.

Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin.

A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection.