Paolo Mereghetti

Brownian Dynamics Simulation of Protein Solutions: Structural and Dynamical Properties

The study of solutions of biomacromolecules provides an important basis for understanding the behavior of many fundamental cellular processes, such as protein folding, self-assembly, biochemical reactions, and signal transduction. Here, we describe a Brownian dynamics simulation procedure and its validation for the study of the dynamic and structural properties of protein solutions. In the model used, the proteins are treated as atomically detailed rigid bodies moving in a continuum solvent. The protein-protein interaction forces are described by the sum of electrostatic interaction, electrostatic desolvation, nonpolar desolvation, and soft-core repulsion terms. The linearized Poisson-Boltzmann equation is solved to compute electrostatic terms. Simulations of homogeneous solutions of three different proteins with varying concentrations, pH, and ionic strength were performed. The results were compared to experimental data and theoretical values in terms of long-time self-diffusion coefficients, second virial coefficients, and structure factors. The results agree with the experimental trends and, in many cases, experimental values are reproduced quantitatively. There are no parameters specific to certain protein types in the interaction model, and hence the model should be applicable to the simulation of the behavior of mixtures of macromolecules in cell-like crowded environments.

The Shape of Protein Crowders is a Major Determinant of Protein Diffusion

As a model for understanding how molecular crowding influences diffusion and transport of proteins in cellular environments, we combined experimental and theoretical approaches to study the diffusion of proteins in highly concentrated protein solutions. Bovine serum albumin and γ-Globulin were chosen as molecular crowders and as tracers. These two proteins are representatives of the main types of plasma protein and have different shapes and sizes. Solutions consisting of one or both proteins were studied. The self-diffusion coefficients of the fluorescently labeled tracer proteins were measured by means of fluorescence correlation spectroscopy at a total protein concentration of up to 400 g/L. γ-Globulin is found to have a stronger influence as a crowder on the tracer self-diffusion coefficient than Bovine serum albumin. Brownian dynamics simulations show that the excluded volume and the shape of the crowding protein have a significantly stronger influence on translational and rotational diffusion coefficients, as well as transient oligomerization, than hydrodynamic or direct interactions. Anomalous subdiffusion, which is not observed at the experimental fluorescence correlation spectroscopy timescales (>100 μs), appears only at very short timescales (<1 μs) in the simulations due to steric effects of the proteins. We envision that the combined experimental and computational approach employed here can be developed to unravel the different biophysical contributions to protein motion and interaction in cellular environments by systematically varying protein properties such as molecular weight, size, shape, and electrostatic interactions.

Diffusion and association processes in biological systems: theory, computation and experiment

Macromolecular diffusion plays a fundamental role in biological processes. Here, we give an overview of recent methodological advances and some of the challenges for understanding how molecular diffusional properties influence biological function that were highlighted at a recent workshop, BDBDB2, the second Biological Diffusion and Brownian Dynamics Brainstorm.

Fourier transform infrared spectroscopy as a method to study lipid accumulation in oleaginous yeasts

BackgroundOleaginous microorganisms, such as different yeast and algal species, can represent a sustainable alternative to plant oil for the production of biodiesel. They can accumulate fatty acids (FA) up to 70% of their dry weight with a predominance of (mono)unsaturated species, similarly to what plants do, but differently from animals. In addition, their growth is not in competition either with food, feed crops, or with agricultural land.Despite these advantages, the exploitation of the single cell oil system is still at an early developmental stage. Cultivation mode and conditions, as well as lipid extraction technologies, represent the main limitations. The monitoring of lipid accumulation in oleaginous microorganisms is consequently crucial to develop and validate new approaches, but at present the majority of the available techniques is time consuming, invasive and, when relying on lipid extraction, can be affected by FA degradation.ResultsIn this work the fatty acid accumulation of the oleaginous yeasts Cryptococcus curvatus and Rhodosporidium toruloides and of the non-oleaginous yeast Saccharomyces cerevisiae (as a negative control) was monitored in situ by Fourier Transform Infrared Spectroscopy (FTIR). Indeed, this spectroscopic tool can provide complementary information to those obtained by classical techniques, such as microscopy, flow cytometry and gas chromatography. As shown in this work, through the analysis of the absorption spectra of intact oleaginous microorganisms it is possible not only to monitor the progression of FA accumulation but also to identify the most represented classes of the produced lipids.ConclusionsHere we propose FTIR microspectroscopy - supported by multivariate analysis - as a fast, reliable and non invasive method to monitor and analyze FA accumulation in intact oleaginous yeasts. The results obtained by the FTIR approach were in agreement with those obtained by the other classical methods like flow cytometry and gas chromatography. Moreover, the possibility to track lipid production in real time is highly desirable to support the initial screening of strains and media as well as to optimize the scaling up experiments, which are essential for a viable and successful development of an industrial production process.

Diffusion of hydrophobin proteins in solution and interactions with a graphite surface

BackgroundHydrophobins are small proteins produced by filamentous fungi that have a variety of biological functions including coating of spores and surface adhesion. To accomplish these functions, they rely on unique interface-binding properties. Using atomic-detail implicit solvent rigid-body Brownian dynamics simulations, we studied the diffusion of HFBI, a class II hydrophobin from Trichoderma reesei, in aqueous solution in the presence and absence of a graphite surface.ResultsIn the simulations, HFBI exists in solution as a mixture of monomers in equilibrium with different types of oligomers. The oligomerization state depends on the conformation of HFBI. When a Highly Ordered Pyrolytic Graphite (HOPG) layer is present in the simulated system, HFBI tends to interact with the HOPG layer through a hydrophobic patch on the protein.ConclusionsFrom the simulations of HFBI solutions, we identify a tetrameric encounter complex stabilized by non-polar interactions between the aliphatic residues in the hydrophobic patch on HFBI. After the formation of the encounter complex, a local structural rearrangement at the protein interfaces is required to obtain the tetrameric arrangement seen in HFBI crystals. Simulations performed with the graphite surface show that, due to a combination of a geometric hindrance and the interaction of the aliphatic sidechains with the graphite layer, HFBI proteins tend to accumulate close to the hydrophobic surface.

Near native-state conformational landscape of psychrophilic and mesophilic enzymes: probing the folding funnel model.

In recent years, increased interest has been directed to the study of enzyme adaptation to low temperatures. In particular, a peculiar folding funnel model was proposed for the free energy landscape of a psychrophilic alpha-amylase and other cold-adapted enzymes. In the present contribution, the comparison between the near native-state dynamics and conformational landscape in the essential subspace of different cold-adapted enzymes with their mesophilic counterparts, as obtained by more than 0.1 micros molecular dynamics simulations at different temperatures, allows the folding funnel model to be probed. Common characteristics were highlighted in the near native-state dynamics of psychrophilic enzymes belonging to different enzymatic families when compared to the mesophilic counterparts. According to the model, a cold-adapted enzyme in its native-state consists of a large population of conformations which can easily interconvert and result in high structural flexibility.

FTIR spectral signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence.

Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050-2800cm(-1)) and protein (1700-1600cm(-1)) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800cm(-1). The use of the multivariate principal component-linear discriminant analysis (PCA-LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data.

Two Interconvertible Folds Modulate the Activity of a DNA Aptamer Against Transferrin Receptor

Thanks to their ability to recognize biomolecular targets with high affinity and specificity, nucleic acid aptamers are increasingly investigated as diagnostic and therapeutic tools, particularly when their targets are cell-surface receptors. Here, we investigate the relationship between the folding of an anti-mouse transferrin receptor DNA aptamer and its interaction with the transferrin receptor both in vitro and in living cells. We identified and purified two aptamer conformers by means of chromatographic techniques. Fluorescence-anisotropy measurements showed that only one fold is able to bind mouse transferrin receptor. Besides displaying enhanced endocytosis in living mouse fibroblasts, the purified active fold is internalized also in human pancreatic cancer cells. Starting from these observations, we rationally designed variations of the parent sequence aimed at stabilizing the active fold, and consequently increase aptamer activity. A truncated version and full-length mutants with higher affinity than the parent sequence are shown.

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes

© 2012 Ami et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes

FT-IR spectroscopy supported by PCA–LDA analysis for the study of embryonic stem cell differentiation

As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a power- ful tool in stem cell research. In this work we characterized in situ by FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differen- tiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA-LDA) that enabled us to segregate ES cell spectra into well sep- arate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050-2800 cm −1 ), protein (1700-1600 cm −1 ) and in the nucleic acid (1050-850 cm −1 ) absorption regions were observed between days 4 to 7 of dif- ferentiation, indicating the appearance - at day 7 - of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm −1 and 899 cm −1 ) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm −1 shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research.