Antonio Emanuele

SELF-ASSEMBLY OF BIOPOLYMERIC STRUCTURES BELOW THE THRESHOLD OF RANDOM CROSS-LINK PERCOLATION

Self-assembly of extended structures via cross-linking of individual biomolecules often occurs in solutions at concentrations well below the estimated threshold for random cross-link percolation. This requires solute-solute correlations. Here we study bovine serum albumin. Its unfolding causes the appearance of an instability region of the sol, not observed for native bovine serum albumin. As a consequence, spinodal demixing of the sol is observed. The thermodynamic phase transition corresponding to this demixing is the determinative symmetry-breaking step allowing the subsequent occurrence of (correlated) cross-linking and its progress up to the topological phase transition of gelation. The occurrence of this sequence is of marked interest to theories of spontaneous symmetry-breaking leading to morphogenesis, as well as to percolation theories. The present results extend the validity of conclusions drawn from our previous studies of other systems, by showing in one single case, system features that we have hitherto observed separately in different systems. Time-resolved experimental observations of the present type also bring kinetic and diffusional processes and solute-solvent interactions into the picture of cross-link percolation.

Irreversible formation of intermediate BSA oligomers requires and induces conformational changes.

Understanding the relation between protein conformational changes and aggregation, and the physical mechanisms leading to such processes, is of primary importance, due to its direct relation to a vast class of severe pathologies. Growing evidence also suggests that oligomeric intermediates, which may occur early in the aggregation pathway, can be themselves pathogenic. The possible cytotoxicity of oligomers of non‐disease‐associated proteins adds generality to such suggestion and to the interest of studies of oligomer formation. Here we study the early stages of aggregation of Bovine Serum Albumin (BSA), a non pathogenic protein which has proved to be a useful model system. Dynamic light scattering and circular dichroism measurements in kinetic experiments following step‐wise temperature rises, show that the “intermediate” form, which initiates large‐scale aggregation, is the result of structural and conformational changes and concurrent formation of oligomers, of average size in the range of 100–200 Å. Two distinct thresholds are observed. Beyond the first one oligomerization starts and causes partial irreversibility of conformational changes. Beyond the second threshold, additional secondary structural changes occurring in proteins being recruited progress on the same time scale of oligomerization. The concurrent behavior causes a mutual stabilization of oligomerization, and of structural and conformational changes, evidenced by a progressive increase of their irreversibility. This process interaction appears to be pivotal in producing irreversible oligomers. Proteins 2004;9999:000–000.

Effect of T-R conformational change on sickle-cell hemoglobin interactions and aggregation.

We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle‐cell hemoglobin (HbS), the β6 Glu‐Val point mutant of adult human hemoglobin (HbA), in its R (CO‐liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points TS(c) by extrapolation. These points were fitted to theoretical expressions of the TS(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model‐free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/mol and 314 J/°K‐mol, respectively. Aggregation was observed also for R‐HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter ϵ = (T − TS)/TS. Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R‐HbS, T‐HbS, and T‐HbA. The distinct effects of their specific nature and configurations, and those of desolvation on the demixing and aggregation thermodynamics, and on the aggregate structure are highlighted. Proteins 2005.

Effects of solvent perturbation on gelation driven by spinodal demixing.

We study effects of solvent perturbation on kinetic competition between spinodal demixing and gelation in agarose solutions at a concentration of 5 g/l. Two different cosolutes (tert-butyl alcohol and trimethyl amine N-oxide) known for altering in opposite way solvent-mediated interactions are chosen. By rheometry, static and dynamic light scattering experiments, we show that the cosolute presence shifts the boundary of the instability region of solution leaving unaffected temperature and polymer concentration values required for percolation. Results suggest that an appropriate choice of quenching temperature and solvent allows controlling the gelation time and the gel structural properties.

Early stages of β2‐microglobulin aggregation and the inhibiting action of αB‐crystallin

Static and dynamic light scattering experiments on extremely clean (nanofiltered) samples of the well‐known amyloidogenic protein β2‐microglobulin (R3Aβ2m and WTβ2m) evidence the self‐assembly of early aggregates showing unexpected features. Further, we find that αB‐crystallin effectively inhibits aggregation of β2m in a far less than stoichiometric proportion, from 1:60 αB‐crystallin monomer to β2m monomer ratio, down to at least a 1:2 × 103 αB‐crystallin oligomerto β2m monomer ratio. Therefore, inhibition of the early stage of β2m aggregation by αB‐crystallin does not necessarily require a mechanicistic chaperon‐like action implying one‐to‐one binding. This highlights the role of the free energy landscape of the system and of related modifications of solute–solvent thermodynamics caused by co‐solutes, in agreement with recent work from our and other laboratories. Proteins 2008.

Y:BaZrO3 perovskite compounds II: designing protonic conduction by using MD models.

The proton dynamics in Y-doped BaZrO(3) derivatives, in particular the different dopant environments within a Pm3m cubic framework, were studied by using classical molecular dynamics (MD) calculations. Single- and double substitution of zirconium by yttrium atoms was considered. The presence of yttrium induced variations in the surrounding oxygen sites, according to their local geometrical arrangements. The differences among such distinct oxygen sites became evident when protons interacted with them and upon changes in the temperature. So, different proton transfer pathways, which had different energy barriers, characterized the topologically different oxygen sites. The experimental proton-hopping activation energy was only reproduced in those structures in which two yttrium atoms formed a Y-O-Y arrangement, which also acted as multilevel protonic traps. Protonic conduction in these materials could be improved by avoiding such yttrium clustering, hence preventing the formation of the protonic traps.

Cation environment of BaCeO3-based protonic conductors II: new computational models.

Quantum chemical calculations have been carried out to simulate Y-doped BaCeO(3) derivatives. Hartree-Fock energy functional was used to study octahedral site environments embedded in a Pmcn orthorhombic framework, showing local arrangement characterized by Ce-O-Ce, Ce-O-Y, and Y-O-Y (Z-O-Ξ) configurations and including or not hydrogen close to the moieties encompassing those configurations. The latter are, in fact, representative of - and, in our modeling approach, were treated as - local arrangements that could be found in Y:BaCeO(3)-doped materials. The geometrical optimizations performed on the structural models and a detailed orbital analysis of these systems allowed us to confirm and deepen new interpretations, concerning experimental findings already reported in the literature. In particular, the bimodal distribution characterizing the Y-O first coordination shell, found by EXAFS analysis, could be attributed to a local clustering of Y atoms showing characteristic Y-O-Y arrangements. Moreover, the local charge analysis, characterizing the models containing or not hydrogen atoms, showed that the moving protons are able to dynamically change the properties of their near environment, in any case, leaving unaltered the global protonic conduction features of the material, irrespective of the kind of cation in a given Z-O-Ξ moiety.

Blowing up reversibility in biomolecular self-assembly

The self-assembly of hydrogels involves the interplay of many processes: thermodynamic demixing, molecular crosslinking and molecular conformational changes. Interactions among these processes lead to complex structures encompassing many length-scales. Here, we monitor experimentally changes of the structure of biomolecular aggregates during the melting of an agarose gel, that is during the inverse process of gelation, which exhibits a large hysteresis. Observations on different lengthscales clarify the role of the processes involved in sustaining irreversibility.