"Complex biological structures solved by small-angle scattering: quaternary structure of proteins and porous defects in membranes".
Prof. Dr. Francesco Spinozzi 

Università Politecnica delle Marche
Data: 01/11/2013
Hora: 15h
Local: Auditório Adma Jafet - Instituto de Física da USP

New methods, recently developed in the molecular biophysics laboratory at the Polytechnic University of Marche (Ancona, Italy), to analyse small-angle X-ray and neutron scattering (SAXS and SANS) data from complex biological structures are presented.
The first method, called QUAFIT, is focused on the determination of the quaternary structure of proteins [1,2]. The method is based on the idea that asymmetric monomers, formed by rigid domains of known structure, possibly connected by flexible linkers of known sequence, are assembled according to a point group symmetry. Scattering amplitudes of domains and linkers are combined by means of a spherical harmonics expansion. In order to avoid any overlap among domains, the “contact distance” between two domains is determined as a function of orientation by a novel algorithm, based on a Stone’s invariants. QUAFIT has been tested by studying the structure of hemocyanin from Octopus vulgaris, a protein that shows a hierarchical organization of monomers. In the first QUAFIT study [1], the structures of the decamer and of the dissociated “loose” monomer have been identified by analysing SAS curves in the most and the least aggregative conditions, respectively. Results are in very good agreement with the structural model derived from electron microscopy observations. Afterwards, other SAS curves corresponding to different chemical-physical conditions have been analysed through QUAFIT, by considering heterogeneous mixtures composed of the entire decamer, the dissociated “loose” monomer and all the intermediate dissociation products. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, appear to be intrinsically heterogeneous [2].

Other methods have been developed to investigate by SAXS structural aspects of a pure anionic phospholipid, dimyristoyl phosphatidyl glycerol (DMPG), during the gel-fluid transition of the alkyl chains. The melting process, which depends on pH, ionic strength, and DMPG concentration, was investigated in aqueous dispersions, using several experimental techniques, with a proposal of pore formation [3, 4]. At DMPG concentrations higher than 70 mM the melting regime was investigated by small (SAXS) and wide-angle (WAXS) X-ray scattering, differential scanning calorimetry, and polarized optical microscopy [5]. A pore model was developed and was able to explain SAXS curves with 70 mM, attributed to in-plane correlation between pores [5]. The lamellar phase presents an abnormal behavior of the intensity of the bilayer band, and for its analysis a model of water-penetrated bilayers was developed [6]. Recently a modified SAXS model has been developed, where the surface of the bilayer is distinct in two planar regions, with chains in the gel and in the fluid conformation, respectively, and in two bent regions, due to presence of large and small pores, both described by a toroidal geometry. For the four regions, the profile of the electron density in the direction perpendicular to the interface between the polar heads of the lipids and the water medium is described by the canonical model of three-density level, corresponding to polar heads, methylene chains and terminal methyl groups. The Fourier transform of the effective electron density is used to interpret the SAXS signal at high values of the transferred momentum. Using this model a global fit of 22 SAXS curves in the temperature interval ~ 19ºC to 40ºC was able to give results on the pore surface fractions and on the several electron densities parameters.
[1] F. Spinozzi and M. Beltramini, Biophys. J., 103:511–521 (2012).
[2] F. Spinozzi, P. Mariani, I. Mičetić, C. Ferrero, D. Pontoni, and M. Beltramini, PLOS one, e49644 (2012).
[3] K.A. Riske, L.Q. Amaral, H.G. Dobereiner and M.T. Lamy, Biophys. J. 86, 3722–3733 (2004).
[4] K.A. Riske, L.Q. Amaral, M.T. Lamy, Langmuir 25, 10083–10091 (2009).
[5] F. Spinozzi, L. Paccamiccio, P. Mariani and L.Q. Amaral, Langmuir 26, 6484–6493 (2010)
[6] F. Spinozzi, P. Mariani, L. Paccamiccio and L.Q. Amaral, J. Phys.: Conf. Ser. 247 012019 (2010).

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