Manno, M.

Martorana, V.

Raccosta, S.

RACCOSTA, Samuele

Martorana, V

Archivio istituzionale della ricerca - Università di Palermo

Role of electrostatic interactions in the fibrillogenesis of  lysozymeSamuele Raccosta1,2, Vincenzo Martorana1 and Mauro Manno11. Institute of Biophysics at Palermo, National Research Council of Italy, Via U. La Malfa 153, 90146 Palermo, Italy.           2. Department of Physical and Astronomical Sciences, University of Palermo, Via Archirafi 36, 90123 Palermo, Italy.    AIM: Highlighting the role of repulsive electrostatic interactionsin protein unfolding and self-assembly, with a focus on theformation of elongated fibrillar aggregates.AFM. Typical size of fibrils is 20 nm inwidth, few microns in length, 50 nm in axialperiodicity.At 70 °C amorphous aggregates are incompetition with fibrils.Amorphous aggregation is enhanced if somesalt (20-200 mM) is added in solution. Ifincubation temperature is lower fibrils areformed even if salt is added [Hill et al. 2009]XV School of Pure and Applied Biophysics, Jan 24-28, 2011 - Venice, Italy                                       Contact: samuele.raccosta@pa.ibf.cnr.it2 22 ( ) 1 ( )Dq tg D P D e dt    -LAG PHASE:Coexistence of Monomers (Rh≈1nm) andOligomers (Rh≈10nm)AFTER A FEW DAYSAppearence of Fibrils and other big aggregates (Rh > 80 nm)Intensity autocorrelation functions g2(t)diffusion coefficient distribution P(D) High protein charge reduces protein stability towards unfolding. Self-assembly at high T due to the exposure of hydrophobic residues is slowed down bythe strong electrostatic repulsive interaction more stable solution and organizedaggregation. The coexistence of monomers and oligomers suggests a competing effect of hydrophobicand electrostatic interaction The kinetics of fibril formation, their morphology and recent FTIR results [Freire et al.2009] suggest that oligomers may be on-pathway fibril precursor. No relevant secondary mechanisms of fibrillationFibrillation kinetics by Static and Dynamic Light ScatteringM0= 14.3 kDa4 S-S bondsZ= +17 e (pH=2)Z= +7.5 e (pH=7)Hydrodynamic diameter 10n [nm]Time[days]Slow conformational changes (60–65 °C): two-state transitionSecondary structure:loss of α-structrure (isodichroicpoint at 204 nm)1) Study more deeply of the protein-protein interactions at different temperaturesand incubation times by SAXS2) Characterization of structure by SAXSTertiary structure:red-shidt of TRP emission(isosbestic point at 368 nm)70°Cdil. 1:10000 Thermal stability at acidic pHAbove 45 °C lysozyme changes conformation reversibly .DSCpH=7: lysozyme is stabilized by surfacesalt bridges.pH=2: all protonable residues are positive, salt bridges are broken,intramolecular repulsive interactions destabilize lysozyme.CD and trp-PL spectra show respectively thedecrease of α-structure [Arnaudov et al. 2005]and the concomitant exposure of trp to thesolvent above 60 °C.Intermolecular interactions at high temperaturea) Experimental data: compressibility curves at differenttemperatures.b) 1st result: Swelling of lysozyme at high temperature.c) 2nd result: Second virial coefficient (B2) has no dependence upontemperature.Lysozyme as a charged sphere with counterions collapsed on itssurface (Manning condensation).Order of magnitude of B2 is explained by a screened intermolecularelectrostatic repulsion.Model system:Hen Egg-White Lysozymenative monomerunfoldedmonomeroligomerfibrilTSecond virial coefficientEffective hydrodynamicradius65 °C200 mM NaCl

Role of electrostatic interactions in the fibrillogenesis of lysozyme

https://iris.unipa.it/retrieve/handle/10447/53042/26176/Venezia2010_raccosta2.pdf

Role of electrostatic interactions in the fibrillogenesis of lysozyme

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Archivio istituzionale della ricerca - Università di Palermo