Biophysical Chemistry

Current research reports and chronological list of recent articles.


The scientific journal Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.

The publisher is Elsevier. The copyright and publishing rights of specialized products listed below are in this publishing house. This is also responsible for the content shown.

To search this web page for specific words type "Ctrl" + "F" on your keyboard (Command + "F" on a Mac). Then: type the word you are searching for in the window that pops up!

Additional research articles see Current Chemistry Research Articles. Magazines with similar content (biophysical chemistry):

 - Biomacromolecules.

 - Faraday Discussions.

 - Journal of Physical Chemistry B.

 - Physical Chemistry Chemical Physics PCCP.



Biophysical Chemistry - Abstracts



Impact of molecular flexibility on the site energy shift of chlorophylls in Photosystem II

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Daniele Narzi, Emanuele Coccia, Marco Manzoli, Leonardo Guidoni

Light harvesting from the Sun by antenna complexes surrounding the reaction center of Photosystem II represents the first step of the natural oxygenic photosynthesis performed by plants, algae and cyanobacteria. The excitation energy derived from the sunlight is absorbed by the chlorophylls of the antenna and subsequently conveyed to the reaction center of Photosystem II through resonant energy transfer mechanisms. In the special pair of chlorophylls of the reaction center the charge separation occurs, eventually leading to the oxidation of water molecules into protons, electrons and molecular oxygen. The adsorption properties of the antenna chlorophylls are ad hoc modulated by the protein environment to guarantee fast energy transfer and minimize side and back reactions. At the same time these properties are influenced by the molecular fluctuations occurring at physiological temperature. In the present work, combining classical molecular dynamics simulations with the Charge Density Coupling method, we estimated the impact of the thermal fluctuations on the site energy shift of the chlorophylls embedded in the Photosystem II complex. Our results show how the effect of the molecular fluctuations is not homogeneous throughout the complex, although the symmetry of the homodimer is maintained. Many peripheral chromophores undergo fluctuations larger then 10kJ/mol around the average values. Possible physiological roles of such fluctuations are discussed.

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Datum: 25.09.2017


Plasma assisted surface treatments of biomaterials

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): L. Minati, C. Migliaresi, L. Lunelli, G. Viero, M. Dalla Serra, G. Speranza

The biocompatibility of an implant depends upon the material it is composed of, in addition to the prosthetic device's morphology, mechanical and surface properties. Properties as porosity and pore size should allow, when required, cells penetration and proliferation. Stiffness and strength, that depend on the bulk characteristics of the material, should match the mechanical requirements of the prosthetic applications. Surface properties should allow integration in the surrounding tissues by activating proper communication pathways with the surrounding cells. Bulk and surface properties are not interconnected, and for instance a bone prosthesis could possess the necessary stiffness and strength for the application omitting out prerequisite surface properties essential for the osteointegration. In this case, surface treatment is mandatory and can be accomplished using various techniques such as applying coatings to the prosthesis, ion beams, chemical grafting or modification, low temperature plasma, or a combination of the aforementioned. Low temperature plasma-based techniques have gained increasing consensus for the surface modification of biomaterials for being effective and competitive compared to other ways to introduce surface functionalities. In this paper we review plasma processing techniques and describe potentialities and applications of plasma to tailor the interface of biomaterials.

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Datum: 25.09.2017


Biochemical and biophysical properties of positively supercoiled DNA

Publication date: Available online 1 September 2017
Source:Biophysical Chemistry

Author(s): Yingting Liu, Andrea M. Berrido, Zi-Chun Hua, Yuk-Ching Tse-Dinh, Fenfei Leng

In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (−) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (−) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (−) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (−) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (−) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (−) sc DNA.

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Datum: 25.09.2017


High-contrast Brillouin and Raman micro-spectroscopy for simultaneous mechanical and chemical investigation of microbial biofilms

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): S. Mattana, M. Alunni Cardinali, S. Caponi, D. Casagrande Pierantoni, L. Corte, L. Roscini, G. Cardinali, D. Fioretto

Mechanical mapping with chemical specificity of biological samples is now made possible by joint micro-Brillouin and micro-Raman measurements. In this work, thanks to the unprecedented contrast of a new tandem Fabry-Perot interferometer, we demonstrate simultaneous detection of Brillouin and Raman spectra from different Candida biofilms. Our proof-of-concept study reveals the potential of this label-free joint micro-spectroscopy technique in challenging microbiological issues. In particular, heterogeneous chemo-mechanical maps of Candida biofilms are obtained, without the need for staining or touching the sample. The correlative Raman and Brillouin investigation evidences the role of both extracellular polymeric substances and of hydration water in inducing a marked local softening of the biofilm.

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Datum: 25.09.2017


Full title pg and editorial board

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229










Datum: 25.09.2017


Nucleocytoplasmic transport in cells with progerin-induced defective nuclear lamina

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Gianmarco Ferri, Barbara Storti, Ranieri Bizzarri

Recent data indicate that nuclear lamina (NL) plays a relevant role in many fundamental cellular functions. The peculiar role of NL in cells is dramatically demonstrated by the Hutchinson-Gilford progeria syndrome (HGPS), an inherited laminopathy that causes premature, rapid aging shortly after birth. In HGPS, a mutant form of Lamin A (progeria) leads to a dysmorphic NL structure, but how this perturbation is transduced into cellular changes is still largely unknown. Owing to the close structural relationship between NL and the Nuclear Pore Complex (NPC), in this work we test whether HGPS affects passive and active nucleo-cytoplasmic shuttling of cargoes by means of an established model based of fluorescence recovery after photobleaching. Our findings clearly demonstrate that dysmorphic NL is decoupled from the dynamic characteristics of passive and active transport towards and from the nucleus, as well as from the binding affinity of transport protein mediators.

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Datum: 25.09.2017


Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Neil A. White, Charles G. Hoogstraten

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2′-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure.

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Datum: 25.09.2017


The effect of β-sheet breaker peptides on metal associated Amyloid-β peptide aggregation process

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): F. Stellato, Z. Fusco, R. Chiaraluce, V. Consalvi, S. Dinarelli, E. Placidi, M. Petrosino, G.C. Rossi, V. Minicozzi, S. Morante

Far-UV Circular Dichroism experiments and Atomic Force Microscopy tomography are employed to assess the impact of β-sheet breakers on the Aβ1–40 peptide aggregation process in the presence of Cu2+ or Zn2+ transition metals. In this work we focus on two specific 5-amino acids long β-sheet breakers, namely the LPFFD Soto peptide, already known in the literature, and the LPFFN peptide recently designed and studied by our team. We provide evidence that both β-sheet breakers are effective in reducing the Aβ1–40 aggregation propensity, even in the presence of metal ions.

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Datum: 25.09.2017


SIBPA under the Tuscan sun: Introduction to the SIBPA XXIII Special Issue

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Daniela Giacomazza, Cristiano Viappiani, Enrico Di Cera, Carlo Musio

The Italian Society for Pure and Applied Biophysics (SIBPA) held its XXIII National Congress in the gorgeous Tuscan town of Cortona, Italy, on September 18-21, 2016. This special issue features a selection of contributions from the Congress in the areas of molecular, applied, cellular and computational biophysics. Cutting-edge developments in nanoscale biophysics were introduced for the first time in the program. SIBPA continues its successful promotion of biophysical disciplines at the national and international levels, with added strength from its partnership with Biophysical Chemistry and Elsevier.

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Datum: 25.09.2017


Building a biomimetic membrane for neutron reflectivity investigation: Complexity, asymmetry and contrast

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Valeria Rondelli, Paola Brocca, Nicola Tranquilli, Giovanna Fragneto, Elena Del Favero, Laura Cantù

The preparation and investigation of model membranes is deserving growing interest both for the physics of complex systems, and for biology. The need of simplified models should preserve mimicking the qualifying characteristics of biological membranes, and keep non-invasive and detailed description. As a main feature, biological membranes are non-homogeneous in the disposition of components, both in the lateral and in the transverse direction. We prepared asymmetric supported membranes containing GM1 ganglioside in biomimetic proportion according to different protocols. Then, we studied their internal structure by neutron reflectometry, providing few-Angstrom sensitivity in the cross direction meanwhile avoiding radiation damage. This technique can also be profitably applied to study interactions at the membrane surface. The best protocol has proven to be the Langmuir-Blodgett/Langmuir-Schaefer depositions. Notably, also the simpler and most accessible protocol of vesicle fusion was found to be suitable for straightforward and good quality deposition of compositionally asymmetric membranes.

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Datum: 25.09.2017


Bioactive compounds from extra virgin olive oils: Correlation between phenolic content and oxidative stress cell protection

Publication date: Available online 23 September 2017
Source:Biophysical Chemistry

Author(s): G. Presti, V. Guarrasi, E. Gulotta, F. Provenzano, A. Provenzano, S. Giuliano, M. Monfreda, M.R. Mangione, R. Passantino, P.L. San Biagio, M.A. Costa, D. Giacomazza

When compared with other edible vegetable oils, the extra virgin olive oil (EVOO) exhibits excellent nutritional properties due to the presence of biophenolic compounds. Although they constitute only a very small amount of the unsaponifiable fraction of EVOO, biophenols strongly contribute to the sensorial properties of this precious food conferring it, for example, the bitter or pungent taste. Furthermore, it has been found that biophenols possess beneficial effects against many human pathologies such as oxidative stress, inflammation, cardiovascular diseases, cancer and aging-related illness. In the present work, the biophenolic content of 51 Italian and Spanish EVOOs was qualitatively and quantitatively identified and their antioxidant ability analyzed by oxygen radical absorbance capacity (ORAC) assay. Results indicated that the maximum relationship can be found if the ORAC value is correlated with the concentration of the large family composed by ligstroside and oleuropein derivatives together with their degradation products, hydroxytyrosol and tyrosol. Then, selected biophenolic extracts were tested in NIH-3T3 cell line to verify their ability in the recovery of the oxidative stress revealed by DCFH-DA assay. Results were linearly correlated with the concentration of ligstroside aglycone (aldehyde and hydroxyl form).

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Datum: 25.09.2017


Full title pg and editorial board

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228










Datum: 25.09.2017


Main photophysical properties of oxyblepharismin

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Barbara Storti, Giovanni Checcucci, Francesco Ghetti, Riccardo Nifosi, Ranieri Bizzarri

Oxyblepharismin is the photo-oxidized form of blepharismin, the chromophore responsible for the photophobic response of heterotrich ciliate Blepharisma japonicum, and represents a nice model for the study of photo-transduction. In this work, we focused on the photophysical characterization of OxyBP, in view of highlighting the main features related to excitation and emission. By a combined experimental and computational approach we identified the main features of absorption and fluorescence emission of the molecule in solvents of different properties, identifying the nature of transitions as well as the possible heterogeneity at ground/excited state. The thorough photophysical characterization of OxyBP is meant to provide the starting point for the elucidation of the photo-transduction pathway in vivo.

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Datum: 25.09.2017


A biophysical approach to quantify skeletal stem cells trans-differentiation as a model for the study of osteoporosis

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): L. Petecchia, F. Viti, F. Sbrana, M. Vassalli, P. Gavazzo

The stroma of human bone marrow contains a population of skeletal stem cells (hBM-MSC) which are common ancestors, among the others, of osteoblasts and adipocytes. It has been proposed that the imbalance between hBM-MSC osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. The possibility to reproduce this mechanism in vitro has been demonstrated, providing a good model to disclose the details of the complex bone-fat generation homeostasis. Nevertheless, the lack of a simple approach to quantitatively assess the actual stage of a cellular population hindered the adoption of this in vitro model. In this work, the direct differentiation of hBM-MSCs towards a single (osteo or adipo) lineage was characterized using quantitative biophysical and biological approaches, together with the parallel process of trans-differentiation from one lineage to the other. The results confirm that the original plasticity of hBM-MSCs is maintained along the initial stages of the differentiation, showing that in vitro conversion of pre-osteoblasts into adipocytes and, vice versa, of pre-adipocytes into osteoblasts is extremely efficient, comparable with the direct differentiation. Moreover, a method based on digital holography is proposed, providing a quantitative indication of the phenotype stage along differentiation.

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Datum: 25.09.2017


Conformational flexibility and base-pairing tendency of the tobacco carcinogen O6-[4-oxo-4-(3-pyridyl)butyl]guanine

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Katie A. Wilson, Kariann G. Szemethy, Stacey D. Wetmore

The present work uses DFT calculations to characterize the conformational and hydrogen-bonding properties of O6-[4-oxo-4-(3-pyridyl)butyl]guanine (POB-G), a DNA adduct caused by tobacco. POB-G is found to adopt many isoenergetic conformations that allow for discrete interactions between the bulky moiety and the adducted G and/or pairing base. The calculated structure and stability of the hydrogen-bonded pairs between the Watson-Crick or Hoogsteen face of POB-G and the canonical DNA nucleobases fully rationalize the previously reported mutational spectra. Specifically, the stable, non-distorted pseudo-Watson-Crick POB-G:T pair explains the predominant GA mutations, while the stable, yet marginally distorted pairs between the Watson-Crick face of POB-G and A or C clarify the GT mutations and non-mutagenic replication. Finally, the stable, yet highly distorted Hoogsteen POB-G:G pair rationalizes the experimentally-observed insertion but lack of persistence of G opposite POB-G in DNA. Overall, these structural insights are critical for guiding future studies that strive to fully understand the adduct mutagenicity, including the accessible conformations and the replication of POB-G-adducted DNA.

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Datum: 25.09.2017


A possible desensitized state conformation of the human α7 nicotinic receptor: A molecular dynamics study

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): L. Chiodo, T.E. Malliavin, L. Maragliano, G. Cottone

The determination of the conformational states corresponding to diverse functional roles of ligand gated ion channels is subject of intense investigation with various techniques, from X-rays structure determination to electrophysiology and computational modeling. Even with a certain number of structures becoming recently available, only few major structural features distinguishing conductive open channel from the non conductive resting protein have been highlighted, while high-resolution details are still missing. The characterization of the desensitized conformation(s) is even more complex, and only few specific characteristics have been identified. Furthermore, experimental data provide conflicting information for different ion channels, adding further complexity to the topic. Desensitization is defined as the transition of the agonist-bound open channel into an ion channel configuration inactive even in the presence of agonists. In this work, we analyze a conformation corresponding to a non conductive state obtained via molecular dynamics simulations of a homology model of the human α7 nicotinic receptor complexed with agonists. We highlight some characteristics that could associate it to a desensitized state. The obtained structure is assessed against experimental data for other ligand gated ion channels that have been putatively associated to active, inactive and desensitized conditions.

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Datum: 25.09.2017


Pathogenic Aβ A2V versus protective Aβ A2T mutation: Early stage aggregation and membrane interaction

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Laura Colombo, Alessio Gamba, Laura Cantù, Mario Salmona, Fabrizio Tagliavini, Valeria Rondelli, Elena Del Favero, Paola Brocca

We investigated the effects of punctual A-to-V and A-to-T mutations in the amyloid precursor protein APP, corresponding to position 2 of Aβ1–42. Those mutations had opposite effects on the onset and progression of Alzheimer disease, the former inducing early AD pathology and the latter protecting against the onset of the disease. We applied Static and Dynamic Light Scattering and Circular Dichroism, to study the different mutants in the early stages of the aggregation process, essential for the disease. Comparative results showed that the aggregation pathways differ in the kinetics and extent of the process, in the size of the aggregates and in the evolution of the secondary structure, resulting in fibrils of different morphology, as seen by AFM. Mutated peptides had comparable toxic effects on N2a cells. Moreover, as assessed by X-ray scattering, all of them displayed disordering effects on the internal structure of mixed phospholipids-gangliosides model membranes.

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Datum: 25.09.2017


Primary cortical neurons on PMCS TiO2 films towards bio-hybrid memristive device: A morpho-functional study

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Alessandro Roncador, Aura Matilde Jimenez-Garduño, Laura Pasquardini, Giovanni Giusti, Nicola Cornella, Lorenzo Lunelli, Cristina Potrich, Ruben Bartali, Lucrezia Aversa, Roberto Verucchi, Mauro Dalla Serra, Silvia Caponi, Salvatore Iannotta, Paolo Macchi, Carlo Musio

We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO2) prepared by Pulsed Microplasma Cluster Source (PMCS). This technique uses supersonic pulsed beams seeded by clusters of the metal oxide synthesized in a plasma discharge. The final stoichiometry of the TiO2 thin films is tuned changing the gas mixture, achieving stoichiometric or oxygen overstoichiometric films. All the films showed consistent biocompatibility and a spontaneous absorption of poly-d-lysine (PDL) that favors the adhesion and growth of murine cortical neurons. Moreover, the bioelectrical activity of the neuronal culture grown on the TiO2 film can be modulated by changing the chemistry of the surface. This work paves the way to develop a bio-hybrid neuromorphic device, where viable nerve cells are grown directly over a titanium dioxide film showing a network of memristors.

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Datum: 25.09.2017


Effect of ionic strength on the aggregation kinetics of the amidated amyloid beta peptide Aβ (1-40) in aqueous solutions

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Adriana Campos-Ramírez, Maripaz Márquez, Liliana Quintanar, Luis F. Rojas-Ochoa

In this work we study the effect of solution ionic strength on the structural evolution of amidated amyloid beta peptide Aβ (1-40) oligomers at the early stages of fibril formation. By light scattering, we follow the time evolution of the structure and short-time dynamics of peptide structures at low ionic strengths. Our results allow identifying initial oligomer structures as the effective building blocks in the amyloid fibrils formation and indicate that the oligomers growth pathway, from compact structures to flexible chain-like structures, becomes faster as the solution ionic strength is increased. Furthermore, we find no evidence of structural branching what suggests that elongation of amyloid fibrils is dominated by linear association. To describe our results we adapt a phenomenological model based on population balance equations and linear polymer growth, where the parameters required are obtained from the experiments. Model calculations are in good agreement with experimentally-obtained estimates for the radius of gyration of Aβ (1-40) oligomers, thus further supporting our findings. Additionally, we introduce a model for the effective interaction among initial Aβ structures that captures the dependence of the effective association rates on solution ionic strength.

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Datum: 25.09.2017


Broadband diffuse optical characterization of elastin for biomedical applications

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Sanathana Konugolu Venkata Sekar, Joo Sin Beh, Andrea Farina, Alberto Dalla Mora, Antonio Pifferi, Paola Taroni

Elastin is a key structural protein of dynamic connective tissues widely found in the extracellular matrix of skin, arteries, lungs and ligaments. It is responsible for a range of diseases related to aging of biological tissues. The optical characterization of elastin can open new opportunities for its investigation in biomedical studies. In this work, we present the absorption spectra of elastin using a broadband (550–1350nm) diffuse optical spectrometer. Distortions caused by fluorescence and finite bandwidth of the laser source on estimated absorption were effectively accounted for in measurements and data analysis and compensated. A comprehensive summary and comparison between collagen and elastin is presented, highlighting distinct features for its accurate quantification in biological applications.

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Datum: 25.09.2017


Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Antonella Battisti, Paola Morici, Francesco Ghetti, Antonella Sgarbossa

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.

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Datum: 25.09.2017


miRNA purification with an optimized PDMS microdevice: Toward the direct purification of low abundant circulating biomarkers

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): G.C. Santini, C. Potrich, L. Lunelli, L. Vanzetti, S.L. Marasso, M. Cocuzza, F.C. Pirri, C. Pederzolli

A reliable clinical assay based on circulating microRNAs (miRNAs) as biomarkers is highly required. Microdevices offer an attractive solution as a fast and inexpensive way of concentrating these biomarkers from a low sample volume. A previously developed polydimethylsiloxane (PDMS) microdevice able to purify and detect circulating miRNAs was here optimized. The optimization of the morphological and chemical surface properties by nanopatterning and functionalization is presented. Surfaces were firstly characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), fluorescamine assay and s-SDTB (sulphosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) assay and subsequently tested for their capacity to adsorb a fluorescent miRNA. From our analysis, modification of surface charge with 0.1% APTMS ((3-Aminopropyl)trimethoxysilane) and 0.9% PEG-s (2-[Methoxy-(polyethyleneoxy)propyl]trimethoxysilane) performed at 60°C for 10min was identified as the best purification condition. Our optimized microdevice integrated with real-time PCR detection, was demonstrated to selectively purify both synthetic and natural circulating miRNAs with a sensitivity of 0.01pM.






Datum: 25.09.2017


Inside front cover: editorial board

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229










Datum: 25.09.2017


“The human use of human beings”: Interdisciplinarity, transdisciplinarity and all that in biophysics and beyond

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Marco Elio Tabacchi, Settimo Termini

Biophysics, just by looking at its name, indicates an interdisciplinary scientific activity, although the notion of interdisciplinarity, as such, seems to be not widely or specifically discussed by biophysicists. The same seems to have happened as well in the early stages of the development of cybernetics, notably in Norbert Wiener's writings. This situation seems to contrast with what has happened in subsequent developments of cybernetics ideas, notably in general system theory and cognitive sciences. After a few general reflections on the notion of interdisciplinarity, its sophisticated variants and the path leading to the birth of cognitive science, we shall refer to Wiener's thought to extracts aspects and indications that could be useful today, also for what concerns the social responsibility of scientists, which could be seen as stemming from a very general form of interdisciplinarity. Highlights: After a few general reflections on the notion of interdisciplinarity, its sophisticated variants and the path leading to the birth of cognitive science, we shall refer to Wiener's thought to extracts aspects and indications that could be useful today, also for what concerns the social responsibility of scientists.

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Datum: 25.09.2017


Compositional analysis of endogenous porphyrins from Helicobacter pylori

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): A. Battisti, P. Morici, G. Signore, F. Ghetti, A. Sgarbossa

Bacteria able to accumulate porphyrins can be inactivated by visible light irradiation thanks to the photosensitizing properties of this class of aromatic pigments (photodynamic therapy, PDT). Since the bacterial resistance to antibiotic is growing, PDT is becoming a valid alternative. In this context, the pathogen Helicobacter pylori (Hp) is a suitable target for PDT since it spontaneously produces and accumulates porphyrins. It is then important to understand the spectroscopic behavior of these endogenous species to exploit them as photosensitizers, thus improving the results given by the application of PDT in the treatment of Hp infections. In this work we extracted porphyrins from both a laboratory-adapted and a virulent strain of Hp, and we performed spectroscopic and chromatographic experiments to collect information about the composition and the spectrophotometric features of the extracts. The main components of the porphyrin mixtures were identified and their relative contribution to the global red fluorescence was examined.

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Datum: 25.09.2017


Interdisciplinarity in Norbert Wiener, a mathematician-philosopher of our time

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Leone Montagnini

The paper focuses on interdisciplinarity in Norbert Wiener looking at his scientific work from a unitary point of view. It begins with a bird's-eye view of the history of the term “interdisciplinarity”, pointing out how the word was the result of a movement of ideas that took place in US science along the whole Twentieth century. This way, the Wiener's conceptions and practices concerning interdisciplinarity are compared with their historical context, showing analogies and peculiarities. For Wiener, interdisciplinary research by very small groups whose members have a very broad interdisciplinary basis is an essential prerequisite for new fundamental ideas for invention and discoveries. On the contrary, in his opinion, mass attacks by large well financed interdisciplinary research groups with a big number of overspecialized member is useful only in a second phase in which invention and discoveries need to be implemented by designers and developers. Finally, through a conceptual matching between Wiener's ideas and the ones of José Ortega y Gasset, it appears how the Wienerian small interdisciplinary group would fit better with the Kuhnian revolutionary phase in science, while the big interdisciplinary group would fit better to the Kuhnian normal science.

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Datum: 25.09.2017


Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.

Publication date: Available online 19 September 2017
Source:Biophysical Chemistry

Author(s): Małgorzata Prokopowicz, Bartosz Greń, Joanna Cieśla, Borys Kierdaszuk

The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed ‘lack of FRET’ phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159–160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λ ex 280nm, 295nm, 305nm and 313nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems.

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Datum: 25.09.2017


Investigation of structural dynamics of Thrombocytopenia Cargeeg mutants of human apoptotic cytochrome c: A molecular dynamics simulation approach

Publication date: Available online 22 September 2017
Source:Biophysical Chemistry

Author(s): Gurusamy Muneeswaran, Subramanian Kartheeswaran, Manickam Pandiaraj, Kaliappan Muthukumar, Muniyandi Sankaralingam, Saravana Vadivu Arunachalam

Naturally occurring mutations to cytochrome c (cyt-c) have been identified recently in patients with mild autosomal dominant thrombocytopenia (low platelet levels), which yield cyt-c mutants with enhanced apoptotic activity. However, the molecular mechanism underlying this low platelet production and enhanced apoptosis remain unclear. Therefore, an attempt is made herein for the first time to investigate the effects of mutations of glycine 41 by serine (G41S) and tyrosine 48 by histidine (Y48H) on the conformational and dynamic changes of apoptotic (Fe3+) cyt-c using all atom molecular dynamics (MD) simulations in explicit water solvent. Our 30ns MD simulations demonstrate considerable structural differences in G41S and Y48H compared to wild type (WT) cyt-c, such as increasing distances between the critical electron transfer residues results in open conformation at the heme active site, large fluctuations in β-turns and α-helices. Additionally, although the β-sheets remain mostly unaffected in all the three cyt-c simulations, the α-helices undergo conformational switch to β-turns in both the mutant simulations. Importantly, this conformational switch of α-helix to β-turn around heme active site should attributes to the loss of intraprotein H-bonds in the mutant simulations especially between NE2 (His26) and O (Pro44) in agreement with the experimental report. Further, essential dynamics analysis reveals that overall motions of WT cyt-c is mainly involved only in the first eigenvector, but in G41S and Y48H the overall motions are mainly in three and two eigenvectors respectively. Overall, the detailed atomistic level information provide a unifying description for the molecular mechanism of structural destabilization, disregulation of platelet formation and enhanced peroxidase activity of the mutant cyt-c's in the pathology of intrinsic apoptosis.

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Datum: 25.09.2017


Investigation on different chemical stability of mitochondrial Hsp60 and its precursor

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Caterina Ricci, Rita Carrotta, Giacoma Cinzia Rappa, Maria Rosalia Mangione, Fabio Librizzi, Pier Luigi San Biagio, Heinz Amenitsch, Maria Grazia Ortore, Silvia Vilasi

In the large class of molecules that maintain protein homeostasis, called molecular chaperones, chaperonins constitute a subclass that specifically assist the correct folding of newly synthesized proteins. Among them, Hsp60 is composed of a double heptameric ring structure with a large central cavity where the unfolded protein binds via hydrophobic interactions and is supported, in this function, by the co-chaperonin Hsp10. Hsp60 is typically located in the mitochondria, but in some pathological situations, such as cancers and chronic inflammatory diseases, Hsp60 accumulates in the cytoplasm. In these cases, cytoplasmatic Hsp60 is a mixture of mitochondrial Hsp60 secreted from mitochondria upon stress, and its precursor, called naïve Hsp60, never entered into the organella. The difference between the naïve and mitochondrial Hsp60s resides in the absence of the mitochondrial import signal (MIS) in the mitochondrial form, but information on their different structure and stability is still lacking. We present here a study on the stability against a chemical denaturant, of the different cytoplasmic Hsp60 species. By combining Circular Dichroism and Small Angle X-ray Scattering as experimental biophysical techniques to investigate Hsp60, we find that naïve and mitochondrial Hsp60 (mtHsp60) forms differ in their stability. Furthermore, specific responses from the two forms are discussed in terms of the biological environment they are working in, thus opening new questions on their biological function.

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Datum: 25.09.2017


Inside front cover: editorial board

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228










Datum: 25.09.2017


A composite polynomial approach for analyzing the indefinite self-association of macromolecules studied by sedimentation equilibrium

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Damien Hall

A different approach is described for analyzing sedimentation equilibrium experiments of indefinitely self-associating systems. The procedure involves application of conservation of mass criteria, along with local evaluation of the weight average molar mass, to generate a polynomial based on a composite pseudo-independent variable. The outlined method does not depend upon non-linear regression to generate a solution, but instead requires evaluation of the roots of a high-order polynomial.

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Datum: 25.09.2017


The isolated, twenty-three-residue-long, N-terminal region of the glutamine synthetase inactivating factor binds to its target

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): José L. Neira, Francisco J. Florencio, M. Isabel Muro-Pastor

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS type I is regulated by protein-protein interactions with a 65-residue-long protein (IF7). IF7 binds initially to GS through residues at its N terminus. In this work, we studied the conformational preferences of the N-terminal region of IF7 (IF7pep, residues Ala7-Ala29), its binding to GS and its functional properties. Isolated IF7pep populated a nascent helix in aqueous solution. IF7pep was bound to GS with an affinity constant of 0.4μM, and a 1:1 stoichiometry. IF7pep did not inactivate GS, suggesting that there were other IF7 regions important to carry out the inactivating function. Binding of IF7pep to GS was electrostatically-driven and it did not follow a kinetic two-state model.

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Datum: 25.09.2017


Controlled single-cell cyclic compression and transcription analysis: A pilot study

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Paola Gavazzo, Loredana Petecchia, Paolo Facci, Massimo Vassalli, Federica Viti

An innovative platform for the study of the molecular mechanisms at the basis of mechanotransduction has been implemented, developing an experimental approach capable of providing controlled dynamic compression stimuli and retrieving the biomolecular response with single-cell sensitivity. The system provides the ability to perform compression-release cycles on single cells with controlled forces in the nN range and a user-defined repetition rate. Experimental procedures to perform qPCR from a small set of single cells were finely tuned. The experimental platform was tested in the context of bone (cell line hFOB 1.19), a physiological environment highly subjected to mechanical stimuli. Target genes were identified in the literature, based on their involvement in the osteogenesis process or in the bone response to mechanical stimuli. qPCR analysis shows an increase in expression of the chosen targets, and confirms the effectiveness of the presented approach for studying living single cells response to dynamic compression.

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Datum: 25.09.2017


Competitive ligands facilitate dissociation of the complex of bifunctional inhibitor and protein kinase

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Taavi Ivan, Erki Enkvist, Hedi Sinijarv, Asko Uri

Dissociation of the complex of a ligand and a protein usually follows the kinetic profile of the first order process and the rate of dissociation is not affected by the presence of competitive ligands. We discovered that dissociation of the complex between a bifunctional ligand and a protein kinase (the catalytic subunit of cAMP-dependent protein kinase), an enzyme possessing 2 different substrate binding sites, was accelerated (facilitated) over 50-fold in the presence of competitive ligands at higher concentrations. Structurally diverse compounds revealed >10-fold different efficiency for acceleration of dissociation of the complex. These results show that the kinetic behavior of flexible biomolecular complexes possessing two spatially separated contact areas is highly dynamic. This property of biomolecular complexes should be carefully considered for effective application of bifunctional ligands for regulation of activity of target proteins in cells.

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Datum: 25.09.2017


In silico analyses of the effects of a point mutation and a pharmacological chaperone on the thermal fluctuation of phenylalanine hydroxylase

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Daichi Hayakawa, Noriyuki Yamaotsu, Izumi Nakagome, Shin-ichiro Ozawa, Tomoki Yoshida, Shuichi Hirono

Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to mutations in phenylalanine hydroxylase (PAH). Recently, small compounds, known as pharmacological chaperones (PhCs), have been identified that restore the enzymatic activity of mutant PAHs. Understanding the mechanism of the reduction in enzymatic activity due to a point mutation in PAH and its restoration by PhC binding is important for the design of more effective PhC drugs. Thermal fluctuations of an enzyme can alter its activity. Here, molecular dynamics simulation show the thermal fluctuation of PAH is increased by introduction of the A313T mutation. Moreover, a simulation using the A313T-PhC complex model was also performed. Thermal fluctuation of the mutant was found to be reduced upon PhC binding, which contributes to restoring its enzymatic activity.

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Datum: 25.09.2017


Dynamical regimes of lipids in additivated liposomes with enhanced elasticity: A field-cycling NMR relaxometry approach

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Carla C. Fraenza, Esteban Anoardo

We study the molecular dynamics of lipids in binary large unilamellar liposomes suspended in D2O composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or soy phosphatidylcholine (SPC) additivated with different percentiles of sodium deoxycholate (SDC). We use the fast field-cycling proton NMR relaxometry technique over a wide timescale and at diverse temperatures. A model previously validated in different formulations is here employed for the relaxometric analysis of elastic vesicles. A new dynamical regime is observed for the first time in additivated DMPC and additivated/non-additivated SPC liposomes. This surprising feature is discussed in terms of vesicle shape fluctuations, enhanced elasticity and lipid & additive diffusion within the membrane. The continuum elastic theory is revisited for a better understanding of recent experiments and those here presented. We address the point of deformability measurements across rigid permeable barriers versus measurements of the bending elastic modulus in free-standing vesicles.

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Datum: 25.09.2017


First moves towards photoautotrophic synthetic cells: In vitro study of photosynthetic reaction centre and cytochrome bc1 complex interactions

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Emiliano Altamura, Rosa Fiorentino, Francesco Milano, Massimo Trotta, Gerardo Palazzo, Pasquale Stano, Fabio Mavelli

Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly “synthetic cells”) can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy. Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles. Next, the details of our ongoing research program are presented. It involves the use of the reaction centre, the coenzyme Q–cytochrome c oxidoreductase, and the ATP synthase for creating an autonomous synthetic cell. We show experimental results on the chemistry of the first two proteins showing that they can efficiently sustain light-driven chemical oscillations. Moreover, the cyclic pattern has been reproduced in silico by a minimal kinetic model.

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Datum: 25.09.2017


Binding kinetics of mutant p53R175H with wild type p53 and p63: A Surface Plasmon Resonance and Atomic Force Spectroscopy study

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Ilaria Moscetti, Anna Rita Bizzarri, Salvatore Cannistraro

The oncogenic mutant p53R175H, one of the most frequently occurring in human cancers and usually associated with poor prognosis and chemo resistance, can exert a dominant negative effect over p53 family members, namely wild type p53, p63 and p73, inhibiting their oncosuppressive function. Novel anticancer strategies based on drugs able to prevent the formation of complexes between p53R175H and the p53 family members call for a deeper knowledge on the molecular mechanisms of their interaction. To this aim, p53R175H/p63 and p53R175H/p53 complexes were investigated in vitro by using Surface Plasmon Resonance and Atomic Force Spectroscopy, two emerging and complementary techniques able to provide interaction kinetic information, in near physiological conditions and without any labelling. Both approaches show that p53R175H forms a very specific and highly stable bimolecular complex with both p63 and p53; with these interactions being characterized by a very high affinity with equilibrium dissociation constant, K D, of about 109 M. These kinetics results, discussed also in connection with those previously reported for the interaction of p53R175H with p73, could inspire the design of suitable anticancer drugs able to antagonize the interaction of p53R175H with the p53 family members, by restoring then their anti-tumour function.

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Datum: 25.09.2017


First observation of metal ion-induced structural fluctuations of α-helical peptides by using diffracted X-ray tracking

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Daiki Usui, Satomi Inaba, Hiroshi Sekiguchi, Yuji C. Sasaki, Toshiki Tanaka, Masayuki Oda

In order to analyze protein structural dynamics, we designed simple model peptides whose structures changed from random-coil to helix-bundle structures by forming stable hydrophobic core in the presence of metal ions. The strategy involved destabilizing a de novo designed three helix-bundle protein by substituting the residues present in its hydrophobic core with histidine and small amino acids. The conformational changes of peptides induced upon binding of Zn2+ to histidine were analyzed using circular dichroism spectroscopy, which revealed peptides, HA and HG, to be good candidates for further analyses. The diffracted X-ray tracking experiments showed that the structural fluctuations of both HA and HG were suppressed upon binding of Zn2+. We succeeded in observing the differences in fluctuations of HA and HG in solution between random-coil like and helix-bundle structures. The metal-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry.

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Datum: 25.09.2017


Molecular dynamics simulations of T-2410 and T-2429 HIV fusion inhibitors interacting with model membranes: Insight into peptide behavior, structure and dynamics

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): I.C.V.C. Mavioso, V.C.R. de Andrade, A.J. Palace Carvalho, A.M.T. Martins do Canto

T-2410 and T-2429 are HIV fusion inhibitor peptides (FI) designed to present a higher efficiency even against HIV strains that developed resistance against other FIs. Similar peptides were shown to interact with model membranes both in the liquid disordered phase and in the liquid ordered state. Those results indicated that such interaction is important to function and could be correlated with their effectiveness. Extensive molecular dynamics simulations were carried out to investigate the interactions between both T-2410 and T-2429 with bilayers of pure 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and a mixture of POPC/cholesterol (Chol) (1:1). It was observed that both peptides interact strongly with both membrane systems, especially with the POPC/Chol systems, where these peptides show the highest number of H-bonds observed so far. T-2410 and T-2429 showed higher extent of interaction with bilayers when compared to T-20 or T-1249 in previous studies. This is most notable in POPC/Chol membranes where, although able to form H-bonds with Chol, they do so to a lesser extent than T-1249 does, the latter being the only FI peptide so far that was observed to form H-bonds with Chol. This behavior suggests that interaction of FI peptides with rigid Chol rich membranes may not be as dependent from peptide/Chol H-bond formation as previous results of T-1249 behavior led to believe. As in other similar peptides, the higher ability to interact with membranes shown by T-2410 and T2429 is probably correlated with its higher inhibitory efficiency.

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Datum: 25.09.2017


Electron current recordings in living cells

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Paolo Trost, Cristiana Picco, Joachim Scholz-Starke, Margherita Festa, Laura Lagostena, Alex Costa, Francesca Sparla, Armando Carpaneto

Living cells exploit the electrical properties of matter for a multitude of fundamental physiological processes, such as accumulation of nutrients, cellular homeostasis, signal transmission. While ion channels and transporters (able to couple ions to various substrates) have been extensively studied, direct measurements of electron currents mediated by specific proteins are just at the beginning. Here, we present the various electrophysiological approaches that have allowed recordings of electron currents and highlight the future potential of such experiments.

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Datum: 25.09.2017


Bohr effect of human hemoglobin: Separation of tertiary and quaternary contributions based on the Wyman equation

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Kehinde Onwochei Okonjo

As a prelude to separating tertiary from quaternary structure contributions to the Bohr effect, we employed the Wyman equation to analyze Bohr data for human hemoglobin to which 2,3-bisphosphoglycerate, 2,3-BPG, is bound. Changes in the pKas of the histidine Bohr groups result in a net reduction of their contributions to the Bohr effect at pH 7.4 compared to their contributions in stripped hemoglobin. The non-histidine 2,3-BPG binding groups - the β-chain terminal amino group and Lys82β - make negative and positive contributions, respectively, to the Bohr effect. The final result is that the Bohr effect at physiological pH is higher for 2,3-BPG bound compared to stripped hemoglobin. Contributions linked to His2β, His77β and His143β enable us to separate tertiary from quaternary Bohr contributions in stripped and in 2,3-BPG bound hemoglobin. Both contributions serve to make the Bohr effect for 2,3-BPG bound hemoglobin higher than for stripped hemoglobin at physiological pH.

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Datum: 25.09.2017


The molten-globule residual structure is critical for reflavination of glucose oxidase

Publication date: Available online 1 September 2017
Source:Biophysical Chemistry

Author(s): Katarína Garajová, Martina Zimmermann, Martina Petrenčáková, Lenka Dzurová, Michal Nemergut, Ľudovít Škultéty, Gabriel Žoldák, Erik Sedlák

Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.

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Datum: 25.09.2017


Rigorous analysis of static light scattering measurements on buffered protein solutions

Publication date: September 2017
Source:Biophysical Chemistry, Volume 228

Author(s): Peter R. Wills, Donald J. Winzor

Attention is drawn to the thermodynamic invalidity of the current practice of analyzing static light scattering measurements on globular proteins in terms of theory for a single solute because of its disregard of the need to consider small species such as buffer components as additional cosolutes rather than as part of the solvent. This practice continues despite its demonstrated inadequacy in studies of sucrose-supplemented protein solutions, where the aberrant behavior was recognized to be a consequence of physical protein interaction with the small cosolute. Failure to take into account the consequences of small cosolute effects renders extremely difficult any attempt to obtain a rigorous thermodynamic characterization of protein interactions by this empirical technique.

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Datum: 25.09.2017


Discovery of a new mexiletine-derived agonist of the hERG K+ channel

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Roberta Gualdani, Maria Maddalena Cavalluzzi, Francesco Tadini-Buoninsegni, Giovanni Lentini

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in the rapid component (IKr) of cardiac action potential repolarization phase. A large number of structurally different compounds block hERG and cause a high risk of arrhythmias. Among the drugs that block hERG channel, a few compounds have been identified as hERG channel activators. Such compounds may be useful, at least in theory, for the treatment of long term QT syndrome. Here we describe a new activator of hERG channel, named MC450. This compound is a symmetric urea, derived from (R)-mexiletine. Using patch-clamp recordings, we found that MC450 increased the activation current of hERG channel, with an EC50 of 41±4μM. Moreover MC450 caused a depolarizing shift in the voltage dependence of inactivation from −64.1±1.2mV (control), to −35.9±1.4mV, whereas it had no effect on the voltage dependence of activation. Furthermore, MC450 slowed current inactivation and the effect of MC450 was attenuated by the inactivation-impaired double mutant G628C/S631C.

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Datum: 25.09.2017


Kinetic stability and sequence/structure studies of urine-derived Bence-Jones proteins from multiple myeloma and light chain amyloidosis patients

Publication date: Available online 1 September 2017
Source:Biophysical Chemistry

Author(s): Luis M. Blancas-Mejía, Emily B. Martin, Angela Williams, Jonathan S. Wall, Marina Ramirez-Alvarado

It is now accepted that the ability of a protein to form amyloid fibrils could be associated both kinetic and thermodynamic protein folding parameters. A recent study from our laboratory using recombinant full-length (encompassing the variable and constant domain) immunoglobulin light chains found a strong kinetic control of the protein unfolding for these proteins. In this study, we are extending our analysis by using urine-derived Bence Jones proteins (BJPs) from five patients with light chain (AL) amyloidosis and four patients with multiple myeloma (MM). We observed lower stability in κ proteins compared to λ proteins (for both MM and AL proteins) in agreement with previous studies. The kinetic component of protein stability is not a universal feature of BJPs and the hysteresis observed during refolding reactions could be attributed to the inability of the protein to refold all domains. The most stable proteins exhibited 3-state unfolding transitions. While these proteins do not refold reversibly, partial refolding shows 2-state partial refolding transitions, suggesting that one of the domains (possibly the variable domain) does not refold completely. Sequences were aligned with their respective germlines and the location and nature of the mutations were analyzed. The location of the mutations were analyzed and compared with the stability and amyloidogenic properties for the proteins in this study, increasing our understanding of light chain unfolding and amyloidogenic potential.

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Datum: 25.09.2017


Sequence-selective binding of C8-conjugated pyrrolobenzodiazepines (PBDs) to DNA

Publication date: Available online 1 September 2017
Source:Biophysical Chemistry

Author(s): Mohammad A. Basher, Khondaker Miraz Rahman, Paul J.M. Jackson, David E. Thurston, Keith R. Fox

DNA footprinting and melting experiments have been used to examine the sequence-specific binding of C8-conjugates of pyrrolobenzodiazepines (PBDs) and benzofused rings including benzothiophene and benzofuran, which are attached using pyrrole- or imidazole-containing linkers. The conjugates modulate the covalent attachment points of the PBDs, so that they bind best to guanines flanked by A/T-rich sequences on either the 5′- or 3′-side. The linker affects the binding, and pyrrole produces larger changes than imidazole. Melting studies with 14-mer oligonucleotide duplexes confirm covalent attachment of the conjugates, which show a different selectivity to anthramycin and reveal that more than one ligand molecule can bind to each duplex.

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Datum: 25.09.2017


Quantifying the influence of 5′-RNA modifications on RNA polymerase I activity

Publication date: Available online 1 September 2017
Source:Biophysical Chemistry

Author(s): Francis D. Appling, Aaron L. Lucius, David A. Schneider

For ensemble and single-molecule analyses of transcription, the use of synthetic transcription elongation complexes has been a versatile and powerful tool. However, structural analyses demonstrate that short RNA substrates, often employed in these assays, would occupy space within the RNA polymerase. Most commercial RNA oligonucleotides do not carry a 5′-triphosphate as would be present on a natural, de novo synthesized RNA. To examine the effects of 5′-moities on transcription kinetics, we measured nucleotide addition and 3′-dinucleotide cleavage by eukaryotic RNA polymerase I using 5′-hydroxyl and 5′-triphosphate RNA substrates. We found that 5′ modifications had no discernable effect on the kinetics of nucleotide addition; however, we observed clear, but modest, effects on the rate of backtracking and/or dinucleotide cleavage. These data suggest that the 5′-end may influence RNA polymerase translocation, consistent with previous prokaryotic studies, and these findings may have implications on kinetic barriers that confront RNA polymerases during the transition from initiation to elongation.

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Datum: 25.09.2017


Altered ionic currents and amelioration by IGF-1 and PACAP in motoneuron-derived cells modelling SBMA

Publication date: October 2017
Source:Biophysical Chemistry, Volume 229

Author(s): Aura M. Jiménez Garduño, Leon J. Juárez-Hernández, María J. Polanco, Laura Tosatto, Daniela Michelatti, Daniele Arosio, Manuela Basso, Maria Pennuto, Carlo Musio

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a motor neuron disease caused by the expansion of a polymorphic CAG tandem repeat encoding a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA is triggered by the binding of mutant AR to its natural ligands, testosterone and dihydrotestosterone (DHT). To investigate the neuronal alterations of motor neuron cell models of SBMA, we applied patch-clamp methods to verify how polyQ expansions in the AR alter cell ionic currents. We used mouse motoneuron-derived MN-1 cells expressing normal AR (MN24Q) and mutant AR (MN100Q treated cells with vehicle EtOH and DHT). We observed a reduction of the current flux mainly at depolarizing potentials in the DHT–treated cells, while the dissection of macroscopic currents showed single different cationic currents belonging to voltage-gated channels. Also, we treated the cells with IGF-1 and PACAP, which have previously been shown to protect MN-1 cells from the toxicity of mutant AR, and we found an amelioration of the altered currents. Our results suggest that the electrophysiological correlate of SBMA is a suitable reference point for the identification of disease symptoms and for future therapeutic targets.

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Datum: 25.09.2017


Two-ion theory of energy coupling in ATP synthesis rectifies a fundamental flaw in the governing equations of the chemiosmotic theory

Publication date: Available online 19 August 2017
Source:Biophysical Chemistry

Author(s): Sunil Nath

The vital coupled processes of oxidative phosphorylation and photosynthetic phosphorylation synthesize molecules of adenosine-5′-triphosphate (ATP), the universal biological energy currency, and sustain all life on our planet. The chemiosmotic theory of energy coupling in oxidative and photophosphorylation was proposed by Mitchell >50years ago. It has had a contentious history, with part of the accumulated body of experimental evidence supporting it, and part of it in conflict with the theory. Although the theory was strongly criticized by many prominent scientists, the controversy has never been resolved. Here, the mathematical steps of Mitchell's original derivation leading to the principal equation of the chemiosmotic theory are scrutinized, and a fundamental flaw in them has been identified. Surprisingly, this flaw had not been detected earlier. Discovery of such a defect negates, or at least considerably weakens, the theoretical foundations on which the chemiosmotic theory is based. Ad hoc or simplistic ways to remedy this defect are shown to be scientifically unproductive and sterile. A novel two-ion theory of biological energy coupling salvages the situation by rectifying the fundamental flaw in the chemiosmotic theory, and the governing equations of the new theory have been shown to accurately quantify and predict extensive recent experimental data on ATP synthesis by F1FO-ATP synthase without using adjustable parameters. Some major biological implications arising from the new thinking are discussed. The principles of energy transduction and coupling proposed in the new paradigm are shown to be of a very general and universal nature. It is concluded that the timely availability after a 25-year research struggle of Nath's torsional mechanism of energy transduction and ATP synthesis is a rational alternative that has the power to solve the problems arising from the past, and also meet present and future challenges in this important interdisciplinary field of research.

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Datum: 25.09.2017






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