A Long Journey into the Investigation of the Structure–Dynamics–Function Paradigm in Proteins through the Activities of the Palermo Biophysics Group

An overview of the biophysics activity at the Department of Physics and Chemistry Emilio Segrè of the University of Palermo is given. For forty years, the focus of the research has been on the protein structure–dynamics–function paradigm, with the aim of understanding the molecular basis of the relevant mechanisms and the key role of solvent. At least three research lines are identified; the main results obtained in collaboration with other groups in Italy and abroad are presented. This review is dedicated to the memory of Professors Massimo Ugo Palma, Maria Beatrice Palma Vittorelli, and Lorenzo Cordone, which were the founders of the Palermo School of Biophysics. We all have been, directly or indirectly, their pupils; we miss their enthusiasm for scientific research, their deep physical insights, their suggestions, their strict but always constructive criticisms, and, most of all, their friendship. This paper is dedicated also to the memory of Prof. Hans Frauenfelder, whose pioneering works on nonexponential rebinding kinetics, protein substates, and energy landscape have inspired a large part of our work in the field of protein dynamics

Deciphering metal-induced oxidative damages on glycated albumin structure and function

Background: Metal ions such as copper or zinc are involved in the development of neurodegenerative pathologies and metabolic diseases such as diabetes mellitus. Albumin structure and functions are impaired following metal- and glucose-mediated oxidative alterations. The aim of this study was to elucidate effects of Cu(II) and Zn(II) ions on glucose-induced modifications in albumin by focusing on glycation, aggregation, oxidation and functional aspects. Methods: Aggregation and conformational changes in albumin were monitored by spectroscopy, fluorescence and microscopy techniques. Biochemical assays such as carbonyl, thiol groups, albumin-bound Cu, fructosamine and amine group measurements were used. Cellular assays were used to gain functional information concerning antioxidant activity of oxidized albumins. Results: Both metals promoted inhibition of albumin glycation associated with an enhanced aggregation and oxidation process. Metal ions gave rise to the formation of β-amyloid type aggregates in albumin exhibiting impaired antioxidant properties and toxic activity to murine microglia cells (BV2). The differential efficiency of both metal ions to inhibit albumin glycation, to promote aggregation and to affect cellular physiology is compared. Conclusions and general significance: Considering the key role of oxidized protein in pathology complications, glycation-mediated and metal ion-induced impairment of albumin properties might be important parameters to be followed and fought. © 2013 Elsevier B.V

Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu2+, Cd2+, Pb 2+ and Ni2+ concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 μM). Moreover, starting from these data, using the combination of molecular biology techniques and optical setup, we developed a device for the detection of Cu2+ in water solutions. The quenching effect detected with the device showed that the relative attenuation of the signal (0.46±0.02 AU) was slightly larger than the data measured by fluorescence spectra (0.65±0.03 AU). The good sensitivity in the span of two orders of the magnitude of Cu2+ concentration, the fact that the instrument is made up of low-cost and sturdy parts and the selective quenching of rAsGFP to copper ions make this setup suited as a low cost, on-the-field, copper ion-specific biosensor. © 2013 Springer Science+Business Media

Properties of Human Hemoglobins with Increased Polarity in the α- or β-Heme Pocket CARBONMONOXY DERIVATIVES

The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A 0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val → Thr mutation has a larger conformational effect on the β-subunits than on the α-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the α-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271–26278). These effects are attributed to a different electrostatic interaction between Oγ of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Oγ in the β-subunit heme pocket

Data concerning the proteolytic resistance and oxidative stress in LAN5 cells after treatment with BSA hydrogels

AbstractProteolytic resistance is a relevant aspect to be tested in the formulation of new nanoscale biomaterials. The action of proteolytic enzymes is a very fast process occurring in the range of few minutes. Here, we report data concerning the proteolytic resistance of a heat-set BSA hydrogel obtained after 20-hour incubation at 60°C prepared at the pH value of 3.9, pH at which the hydrogel presents the highest elastic character with respect to gel formed at pH 5.9 and 7.4 “Heat-and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold” (G. Navarra, C. Peres, M. Contardi, P. Picone, P.L. San Biagio, M. Di Carlo, D. Giacomazza, V. Militello, 2016) [1]. We show that the BSA hydrogel produced by heating treatment is protected by the action of proteinase K enzyme. Moreover, we show that LAN5 cells cultured in presence of BSA hydrogels formed at pH 3.9, 5.9 and 7.4 did not exhibit any oxidative stress, one of the first and crucial events causing cell death “Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases?” (M. Di Carlo, D. Giacomazza, P. Picone, D. Nuzzo, P.L. San Biagio, 2012) [2] “Effect of zinc oxide nanomaterials induced oxidative stress on the p53 pathway” (M.I. Setyawati, C.Y. Tay, D.T. Leaong, 2013) [3]

Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-assembly, with a special focus on the detection of amyloidogenic polymers

Synthesis and characterization of CdS nanoparticles embedded in a polymethylmethacrylate matrix

CdS nanopowder capped with sodium bis(2-ethylhexyl)sulfosuccinate was synthesized by using water-in-oil microemulsions. The CdS nanoparticles of about 5 nm obtained were embedded in polymethylmethacrylate matrix by a photocuring process. The transparent yellow solid compound was characterized by optical absorption and emission spectroscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The properties of this compound were compared with those of the nanopowder dispersed in heptane and in methylmethacrylate. The results obtained indicate that the nanoparticles are homogeneously dispersed in the matrix and do not change in size during the embedding process. Even if the surface slightly changes its luminescence properties, as a consequence of the different new chemical environment, the final product seems to be suitable for practical applications

Thermal induced conformational changes involved in the aggregation of beta-lactoglobulin

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) molecular probe. This technique revealed to be particularly suitable in investigating samples in the low concentration range and at the initial steps of the aggregation process. The Rayleigh scattering of the exciting light follows the growth of the intermolecular interactions at the same time. Complementary information is also obtained by circular dichroism (CD) measurements on samples in the same experimental conditions. The obtained data indicate a well-defined interconversion between quaternary, ternary and secondary structures, together with conformational rearrangements driven by hydrophobic interactions and intermolecular bonds. The results are also discussed in comparison with similar studies on BSA aggregation

Conformational changes involved in thermal aggregation processes of bovine serum albumin

We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the two tryptophans located in two different domains, in way to study conformational changes in the surrounding of these residues. We also follow emission spectra of Fluorescein-5-Maleimide dye bound to the single free cysteine of BSA. Complementary information on the extent of aggregation and on the structural changes is obtained by Rayleigh scattering and circular dichroism measurements. These data contribute to clarify the connection between conformational changes at tertiary and secondary structure level during the aggregation and how the different domains are involved. We also discuss the relevant role played by cysteine 34 in the aggregation pathways. © 2003 Elsevier B.V. All rights reserved