Mirko Busato

Transient Interactions of a Cytosolic Protein with Macromolecular and Vesicular Cosolutes: Unspecific and Specific Effects.

Cytosolic proteins do not occur as isolated but are exposed to many interactions within a crowded cellular environment. We investigated the associations between a test cytosolic protein, human ileal bile acid binding protein (IBABP), and model cosolutes mimicking macromolecular and lipid membrane intracellular components. Using fluorescence spectroscopy, heteronuclear NMR, and molecular dynamics, we found that IBABP associated weakly with anionic lipid vesicles and experienced transient unspecific contacts with albumin. Localized dynamic perturbations were observed even in the case of apparent unspecific binding. IBABP and ubiquitin did not display mutually attractive forces, whereas IBABP associated specifically with lysozyme. A structural model of the IBABP–lysozyme complex was obtained by data‐driven docking simulation. Presumably, all the interactions shown here contribute to modulating functional communication of a protein in its native environment.

Structural modeling of G-protein coupled receptors: An overview on automatic web-servers.

Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well.

Molecularly imprinted polymers coupled to matrix assisted laser desorption ionization mass spectrometry for femtomoles detection of cardiac troponin I peptides

Molecularly imprinted polymers (MIPs) were combined to MALDI‐TOF‐MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7–2μm), dissociation constants (58–817 nM), kinetics of binding (5–60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI‐TOF‐MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP‐SE MALDI‐TOF‐MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP‐SE MALDI‐TOF‐MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes. Copyright

Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.

Computational design of molecularly imprinted polymer for direct detection of melamine in milk

ABSTRACT A novel protocol for use of molecularly imprinted polymer (MIP) in analysis of melamine is presented. Design of polymer for melamine has been achieved using a combination of computational techniques and laboratory trials, the former greatly reducing the duration of the latter. The compatibility and concerted effect of monomers and solvents were also investigated and discussed. Two novel open-source tools were presented which are the online polymer calculator from mipdatabase.com and the application of the Gromacs modelling suite to determine the ideal stoichiometric ratio between template and functional monomer. The MIP binding was characterised for several structural analogues at 1–100 μM concentrations. The use of divinylbenzene (DVB) as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor (IF) of 2.25 for melamine. This polymer was used in high-performance liquid chromatography (HPLC) for the rapid detection of melamine in spiked milk samples with an experimental run taking 7–8 min. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications.

In Silico Synthesis of Synthetic Receptors: A Polymerization Algorithm

Molecularly imprinted polymer (MIP) synthetic receptors have proposed and applied applications in chemical extraction, sensors, assays, catalysis, targeted drug delivery, and direct inhibition of harmful chemicals and pathogens. However, they rely heavily on effective design for success. An algorithm has been written which mimics radical polymerization atomistically, accounting for chemical and spatial discrimination, hybridization, and geometric optimization. Synthetic ephedrine receptors were synthesized in silico to demonstrate the accuracy of the algorithm in reproducing polymers structures at the atomic level. Comparative analysis in the design of a synthetic ephedrine receptor demonstrates that the new method can effectively identify affinity trends and binding site selectivities where commonly used alternative methods cannot. This new method is believed to generate the most realistic models of MIPs thus produced. This suggests that the algorithm could be a powerful new tool in the design and analysis of various polymers, including MIPs, with significant implications in areas of biotechnology, biomimetics, and the materials sciences more generally.

A genetic approach for synthesizing metabolic models from time series

In this paper we introduce a new approach, based on genetic algorithms and multiple linear regression, for the synthesis of flux regulation functions in metabolic models from observed time series. Genetic algorithms are used as a variable selection technique to identify the best primitive functions for flux regulation, and multiple linear regression is employed to compute primitive function coefficients. Our methodology is here successfully applied to synthesize a set of regulation functions able to regenerate an observed dynamics for the mitotic oscillator in early amphibian embryos.

MIRATE: MIps RATional dEsign Science Gateway

Abstract Molecularly imprinted polymers (MIPs) are high affinity robust synthetic receptors, which can be optimally synthesized and manufactured more economically than their biological equivalents (i.e. antibody). In MIPs production, rational design based on molecular modeling is a commonly employed technique. This mostly aids in (i) virtual screening of functional monomers (FMs), (ii) optimization of monomer-template ratio, and (iii) selectivity analysis. We present MIRATE, an integrated science gateway for the intelligent design of MIPs. By combining and adapting multiple state-of-the-art bioinformatics tools into automated and innovative pipelines, MIRATE guides the user through the entire process of MIPs’ design. The platform allows the user to fully customize each stage involved in the MIPs’ design, with the main goal to support the synthesis in the wet-laboratory. Availability: MIRATE is freely accessible with no login requirement at http://mirate.di.univr.it/. All major browsers are supported.