Molecular Biophysics

Molecular Biophysics
Science- 1st Building - Ground Floor
Research Activity
Staff
Equipment
Research Activity
The scientific activities carried on in the Molecular Biophysics Laboratory concern the structural characterization of different molecular systems of biological interest mainly by means of x-rays and neutron Small-Angle  Scattering  and Diffraction techniques. 
Scientists at the Molecular Biophysics Laboratory are frequent users of national and European Large Scale Facilities based on Synchrotron Radiation and Neutron Sources. In particular, European Synchrotron Radiation Facility as well as Institut Laue-Langevin are between our most used facilities. 
Il reattore nucleare dell'Institut Laue-Langevin, Grenoble (F) Esperimenti SAXS al sincrotrone di Amburgo (D)
Main research lines
The main research lines include the self-assembly mechanisms of guanosines and their derivatives and chirality propagation, the analysis of structure and stability of lipid phases, and the structure and aggregation of proteins in solution.
Guanosine self-assembly
In this research line self-assembly processes of guanosine and of its derivatives (lipophilic, too), which form macroaggregates constituted by four-stranded helical filaments (quadruplexes), are investigated. The aggregation of short-chain of double-stranded DNA and single-stranded DNA, which are partially complementary and therefore capable to form nanostars structures with different valence, are object of our research, too. These systems are the subject of an intense theoretical and experimental work, which concerns: 
the structural properties of four-stranded helices; 
the thermodynamic aspects that determine length and stability; 
the mechanism of hierarchical self-assembly in dilute solutions; 
the switching between structural nanoribbons and nanowires observed in organic solvents in the presence and absence of counterions; 
the transmission mechanism of the long-range order; 
the formation of lyotropic liquid-crystalline phases; 
the propagation of chirality; 
the condensation promoted by counterions in aqueous solution; 
the separation of phase in osmolytes and the formation of hydrogels. 
It is worth to note that from a biological point of view, the formation of quadruplexes can be correlated with some biophysical properties of chromatin, with the mechanism of action of telomerase, with some anticancer drugs and even with the source of the prebiotic DNA molecule.
Polimorfismo liotropico del DNA. La molecola è schematizzata come un cilindro. L'immagine è stata ottenuta al microscopio polarizzatore, in un campione di DNA con gradiente di concentrazione Struttura di un quadruplesso formato da GMP sale di potassio. L'immagine è stata ottenuta per diffrazione dei raggi X da un campione di fibre. Self-assembling della GMP: struttura molecolare di un tetramero, un ottamero e un quadruplesso di GMP. La sfera rossa rappresenta uno ione alcalino. Self-assembling di derivati lipofili della guanosina in solventi organici. In assensa di ioni alcalini, gli aggregati sono nastri planari a differente conformazione
Structure and stability of lipid phases
The aspects of lipid systems that we investigate concern: 
the structural characterization of the phases and the lyotropic polymorphism as a function of different experimental parameters (such as, composition, temperature, mechanical pressure, presence of counterions, pH, organic solvents); 
the mechanisms of growth and transformation of macromolecular aggregates; 
the stability of the different phases and of the aggregates themselves (based on analysis of different energy and molecular contributions); 
the hydration properties connected with the self-assembly and to the polymorphic behavior (with particular reference to non-lamellar lipid phases). 
Recently, this research line has increasingly focused on the problem of drug delivery, by studying the structural properties and the thermal stability of lipid nanoparticles exploited as biocompatible and stable drug carriers. The research activities concern the solid lipid nanoparticles, the cubosomes (obtained by sonication after adding suitable surfactants from cubic phases of monoglycerides) and hexosomes (also obtained by sonication from hexagonal phases lipid in the presence of suitable surfactants).
Porous defects in model membranes due to the spontaneous formation of water pores of to the presence of membrane proteins are investigated by analysing SAXS data with new theoretical models based on the possible presence of horizontal correlations among these defects.
Polimorfismo lipidico: diagramma di fase teorico per sistemi rigorsamente liotropici. La struttura delle diverse fasi è mostrata in forma schematica: a,b,c, d, fasi cubiche; H_I e H_II, fasi esagonali diretta e inversa; La, fase lamellare liquido-cristallina. Polimorfismo lipidico: diagramma di fase sperimentale della monooleina in acqua. La figura mostra in forma schematica la struttura delle diverse fasi. Le due fasi cubiche sono bicontinue.  MAD, Monoolein Aqueous Dispersions: immagini al microscopio elettronico di nanoparticelle ottenute per dispersione di fasi cubiche di monooleina in acqua e utilizzate per veicolare farmaci. Gli spettri FFT (mostrati nelle riquadrature) confermano la presenza di cubosomi.
Giant Unilamellar Vesicles

Another particular system investigated in our laboratory belongs to Giant Unilamellar Vesicles (GUVs) with size comparable to that of the cell (diameter 1-100 μM), which can be observed with the optical microscope. GUVs constitute an extremely useful and versatile tool for the in vitro study of the properties of lipid membranes and their interaction with biological macromolecules, since by varying the lipid composition and inserting specific components (such as cholesterol and proteins) the main features of plasma membranes can be easily mimed. At the Molecular Biophysics Laboratory a system for the production of GUVs has been developed, based on the electroporation process in a flow chamber. An optical system (camera and digital camera) for recording images obtained with the polarizing microscope is available. Right now, the research activity concerns the thermal and osmotic stability of GUVs, their behavior as a function of lipid composition or temperature and their interactions with amyloid protofibrils or with amyloid fibrils. In a similar way, the response of liposomes miming cellular membranes to the presence of amyloid fibrils was reported in our paper available here free.

Preparazione di GUV (Giant Unilamellar Vesicle) per elettroformazione con corrente alternata. La cella di crescita è fatta utilizzando due vetrini di  indium tin oxide (ITO)-covered glass appoggiati uno contro l'altro su uno spaziatore di teflon. GUVs di POPC (palmitoil-oleil-fosfatidilcolina). Le immagini al microscopio ottico mostrano come le GUVs inglobino gradualmente le protrusioni generate da fenomeni di budding e pearling. Stabilità termica di GUVs di POPC formate in presenza di fibre amiloidi di citocromo C. Le immagini sono state ottenute al microscopio ottico in funzione della temperatura.
Structure, aggregation and interactions of proteins in solution
This research line concerns the investigation of the structural properties of proteins in solution (including model proteins, proteins not yet crystallized and protein in non-native conditions) by means of small-angle x-rays and neutron scattering (SAXS and SANS, SAS in general) techniques. We study:
• the low resolution shape of proteins;
• the association / dissociation processes;
• the folding / unfolding / misfolding mechanisms induced by the temperature or by the mechanical pressure;
• the protein structure in the presence of co-solvents, as in the case of neuroprotective compounds ;
• the determination of the protein-protein interaction potential.
In the case of proteins not yet crystallized, SAXS or SANS analysis regards the determination of shape, size and aggregation state of the protein of interest, and the evaluation of the conformational changes induced by external agents (such as allosteric inhibitors or activators). In such studies, innovative strategies for data analysis were developed, which allow obtaining more detailed structural and thermodynamic information than those provided by traditional approaches. The developed methods, primarily based on global fitting approaches, are collected in a suite of shareware programs (MPOLE, SASMOL and GENFIT), and validated by the Scientific Software Group of ESRF (Grenoble) and thus included among the scientific software made available to the users of the synchrotrons or nuclear sources.
The quaternary structure of proteins is derived by SAS data with the QUAFIT method, based on the assumption that the protein monomer is constituted by rigid domains of known atomic structure and flexible linkers with known primary sequence.
Recently we are developing new methods to investigate the properties of intrinsically disordered proteins (IDPs) by combining SAXS or SANS measurements with conformational ensembles determined by Molecular Dynamics or Monte Carlo techniques. The misfolding of IDPs leads to the formation of amyloid protofibrils or fibrils, which are the molecular basis of the so-called neuropathologies, such as Alzheimer’s disease and Parkinson’s disease.
Copertina del Biophysical Journal del 6 gennaio 2010, che schematizza i potenziali di interazione tra due proteine, come ottenuti dall'analisi mediante GENFIT di curve SAXS da BSA (l'albumina di siero bovino) in soluzione concentrata. Copertina del Biophysical Journal dell'8 agosto 2012, che schematizza le proprietà strutturali e di aggregazione della emocianina di Octopus Vulgaris in soluizone, come ottenuti dall'analisi mediante QUAFIT di curve SAXS e SANS. Analisi SAXS di proteine in soluzione: la struttura tetramerica della Carbossi Peptidase di Sulfolobus solfataricus in soluzione è confermata dal confronto con la forma ricostruita a partire da curve SAXS mediante MPOLE. Fibre amiloidi preparate a partire da citocromo C in soluzione diluita. L'immagine è ottenuta con un microscopio polarizzatore con Nicol incrociati. Esperimenti SAXS al sincrotrone di Grenoble (ESRF)
Main current research projects 
Currently two national research projects financed by Italian Minister of Instruction, University and Research (MIUR) and one project financed by MIUR via Sincrotrone Elettra for ESS (European Spallation Source) are running:
- PRIN 2010LKE4CC: the project focuses on the study of DNA self-assembly, in order to employ the selectivity of DNA interactions to create new materials with possible application to molecular sensors, to diagnostic and to drug-delivery (Principal Investigator: Paolo Mariani).
- FIRB 2012, RBFR12SIPT: the project focuses on the study of different neuro-protective agents able to influence the aggregation of the amyloid peptide (Aβ), linked to Alzheimer ’s disease, in order to modify the pathogenesis cascade. The founding of agents able to modify Aβ aggregation pattern starts from both chemical-physics studies and from innovative biological/ medical considerations (Principal Investigator and National Coordinator: Maria Grazia Ortore).
- IPHONE: the project basis on the development of an integrated system with a sample-stage able to contain protein solutions and to let simultaneous neutron scattering and circular dichroism experiments. Also time-resolved measurement after stopped-flow mixture is planned (Principal Investigator: Paolo Mariani).
Staff
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071-2204608
071-2204608
Mario Pergolini
Tecnico di Laboratorio
071-2204608
Dott. Caterina Ricci
PhD Student
071-2204608
Dott. Silvia Moscatelli
PhD Student
071-2204608
Dott.ssa Federica Carducci
Phd Student
Dott. Benedetta Come
PhD Student
Dott. Paolo Moretti
PhD Student
Equipment
Experimental techniques
Small Angle X-ray and Neutron Scattering, and X-ray diffraction are the mainly used techniques in our laboratory. X-ray diffraction experiments are performed by using two SAXS camera available in DiSVA, while SAXS and SANS experiments are generally performed in national (Sincrotrone Elettra), European (ILL, ESRF, FMRII, LLB, PETRAIII), and extra-European (LNLS, Brazil) facilities.
Il sincrotrone Europeo ESRF (European Synchrotron Radiation Facility) di Grenoble (F) Le sorgenti di luce Elettra (sincrotrone) e FERMI (free-electron laser) di Trieste
Centrifuge Hermle Z200A for falcon - with rotor
ALC 4214 microcentrifuge
Analytical balance Mettler AE163
Electrophoresis systems BioRad
Pan ultrasound Bransonic
Zeiss stereo microscope
Speedvac for concentration / evaporation
Manufacturer distilled water USF Purelab - Steroglass
Refrigerated incubator Generalcontrol
Scanners for image plates, DenOptix
Polarized light microscope Leitz Ortolux with temperature control and digital camera
Phase contrast microscope Leica DMLS
Generator Philips DY673 monochromator and chamber Guiner with Image Plate
Generator Ital-Structures C3K5 monochromator chamber and Guinier with Image Plate
Digital Camera Nikon D5100
Edwards lyophilizer
System for electroporation and cells with electrodes
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