Giuseppe Briganti

Key role of proximal water in regulating thermostable proteins.

Three homologous proteins with mesophilic, thermophilic and hyperthermophilic character have been studied via molecular dynamics simulations at four different temperatures in order to investigate how water controls thermostability. The water-exposed surface of the protein is shown to increase with the degree of thermophilicity, and the role of water in enhancing the protein internal flexibility and structural robustness is elucidated. The presence of water-water hydrogen bond clusters enveloping the macromolecule is shown to correlate with thermal robustness when going from the mesophilic to the hyperthermophilic variants. Our analysis indicates that essential contributions to thermostability stem from protein-water surface effects whereas the protein internal packing plays a minor role.

Electrical conductivity and dielectric dispersion of E. coli 70S ribosomes and of 30S and 50S subunits: effects of magnesium ions

Abstract Electrical conductivity and dielectric dispersions have been measured on suspensions of 30S, 50S subunits and 70S ribosomal particles from E. coli, in the presence and absence of magnesium ions. Conductivity measurements show a net contribution due to the disperzed phase. Ribosomal particles act in general as obstructants to the ionic motions in the solution, but besides this effect, there is an intrinsic contribution to the overall conductivity which is more pronounced in the absence than in the presence of magnesium ions. This effect suggests the existence of an interfacial conductivity, modulated by bound magnesium ions. The intrinsic conductivity is higher in the 30S subunit than in the 50S. Dielectric dispersions show a consistent difference between 30S and 50S, indicating that the exposure of ribosomal RNA should be greater in the 30S than in the 50S. Structural information is derived from the conductivity measurement, via the Looyenga equation.

Sphere versus Cylinder: The Effect of Packing on the Structure of Nonionic C12E6 Micelles

Two independent series of calculations are performed simulating spherical and cylindrical C12E6 micelles in a temperature range around the experimental sphere-to-rod transition temperature for surfactant concentrations less than 20% by weight. A comparative analysis of these systems helps to shed light on the microscopic details of the micelle sphere-to-rod transition. In agreement with theoretical models, we find that spherical and cylindrical micelles have a different oil core packing; the core radius of a cylindrical micelle is reduced by a factor of 0.87 with respect to the core radius of a spherical micelle. Despite this contraction, the specific volume of the alkyl tails is larger in a cylindrical micelle than in a spherical micelle. In both geometries, this specific volume follows the same linear increase with temperature. Density measurement experiments are also performed in order to evaluate the specific volume of the hydrophobic tail of surfactants of the C12Ej family with j ranging from 5 to 8. We observe a good agreement between experimental data and simulation results. Our simulations also show that the spatial distribution of the head groups in the interface is more effective in screening the oil core in the cylindrical aggregate than in the spherical aggregate, reducing by a factor of 2 the oil surface per monomer exposed to water. This screening accounts for a free-energy difference of Deltafs=fssph-fscyl approximately +2.5kBT per monomer and mirrors the essential role that the hydrophobic interactions have on the shape transition. We also find that the different interface packing correlates with different conformations and flexibility of the hydrophilic fragments E6, that appear as an entropic reservoir for the transition. Finally, comparing the degree of hydration of a spherical micelle at T=283 K with that of a cylindrical micelle at T=318 K, we observe an amount of dehydration in agreement with reported experimental data across the sphere-to-rod transition. However, for aggregates of fixed shape, we find a much smaller amount of dehydration with temperature, suggesting that the shape transition is not a consequence of the measured temperature dehydration but rather the opposite.

Pressure-induced core packing and interfacial dehydration in nonionic C12E6 micelle in aqueous solution.

A spherical micelle of C12E6 is simulated at different pressures, from 0.001 to 3 kbar, by molecular dynamics. On increasing the pressure the alkyl tails of the surfactants pack tightly and stretch. At 3 kbar we observe dynamical slowing down of the oil core of the micelle. At that pressure the core is characterized by a high oil density, rho oil approximately 0.85 g/cm(3), regular density oscillation, and low chain entropy. Pressure affects the interfacial region as well. Dehydration, induced by the collapse of the hydrophilic head groups, is observed in the inner part of the interface. Such dehydration resembles temperature dehydration but differs in details. Our results support the interpretation of recent experiments on micellar solutions at high pressure.

Water around thermophilic proteins: the role of charged and apolar atoms

The thermal response of three proteins with mesophilic, thermophilic and hyperthermophilic character hints at the essential role played in thermostability by the protein-water interface. The formation of spanning water clusters enveloping the macromolecule and their resistance to thermal stress is shown to correlate with the charge distribution at the protein surface; in particular our findings suggest an effective role of the superficial charge distribution in stabilizing the global connectivity of the hydration water.

Anomalous dielectric behavior of undulated lipid membranes. Theoretical model and dielectric spectroscopy measurements of the ripple phase of phosphatidylcholine

We measured the static and dynamic dielectric properties of phosphatidylcholine (PC) spherical membranes (liposomes) over a wide range of temperature and for different liposome radii, pH, and liposomes concentration. Within the investigated temperature range, the physical state of the membrane goes from a gellike to a fluidlike phase, passing through a narrow (≈10 °C) intermediate state, the Ripple phase, characterized by permanent surface undulations of defined wavelength and amplitude. The dielectric properties of the ripple phase are anomalous, the mean static permittivity is higher than that of the gel and fluid phases (where no undulations are present), while the average relaxation frequencies is smaller. Furthermore, the static dielectric permittivity of the fluid phase is much higher than that of the gel phase, while the relaxation frequencies behave just in the opposite way. In order to rationalize this complex behavior we have developed a dynamic mean-field model aimed to calculate the electric p...

Effects of magnesium and temperature on the conformation and reassociation of Escherichia coli and Sulfolobus solfataricus ribosomes

The structural response of the ribosomes of the extremely thermophilic archaeon Sulfolobus solfataricus was analysed and compared to that of the mesophilic (E. coli) ribosomes by assaying ethidium bromide (EB) binding to the native 70S particles as a function of magnesium concentration. We found that the thermophilic ribosomes bound more EB than their mesophilic counterparts; on the other hand, inhibition of EB binding by Mg2+ ions was more effective in the E. coli 70S particle. In Sulfolobus, the separated 30S and 50S subunits and the 70S particle bound the drug in a similar fashion, whereas the E. coli 70S had a reduced number of binding sites with respect to the subunits. Light scattering measurements as a function of Mg2+ concentration were carried out at various temperatures to study the interaction between the ribosomal subunits from the thermophilic and the mesophilic bacteria. As expected, the association of ribosomal subunits in E. coli was magnesium dependent and could be observed also at low temperature. By contrast, the interaction between Sulfolobus ribosomal subunits was obligatorily dependent upon both magnesium ions and a temperature of at least 80 degrees C, close to the physiological optimum for cell growth (87 degrees C).

Dielectric spectroscopy analysis on the interfacial composition of non-ionic micellar solutions

Abstract By iterative use of a suitable mixture equation for the dielectric permittivity of heterogeneous systems we investigate C12E6 solutions as a function of the monomer concentrations and of the temperature at four different solvent compositions: 0, 2, 4 and 6 molar urea in water. With this methodology we obtain the permittivity of the micellar head group crown termination as a function of the hydrated volume of the monomer head group, composed in our case by 6 PEO (polyethylene oxide) units and by the interdigitated solvent molecules. Then the intercept between the dielectric behaviour as a function of the PEO molar concentration of the micellar crown region and of a solution of PEO-water-urea gives an estimate of the actual composition within the interface, at a fixed temperature and solvent composition. Our results suggest a temperature dependence of the degree of hydration at the micellar interface in pure water solution. With mixed water-urea solutions, cosolvent adsorbtion at the micellar interface is observed.

Functional role of Chaperonin protein complexes

Abstract The chaperonins are high-molecular weight protein complexes present in all living cells; they are thought to participate in the folding or refolding of cellular proteins and may also have a function in RNA metabolism. Recently, it has been reported that the chaperonin of thermophilic archeon Sulfolobus solfataricus interacts with the 16S ribosomal RNA and participates in the early stages of its maturation. By means of contrast variation SANS we demonstrate that the native S. solfataricus chaperonin is complexed with a nucleic acid molecule of about 1600 nucleotides, which becomes detached from the protein mojety in the presence of ATP. Treatment with ATP also provokes a conformational change in the protein complex, compacting its structure and closing its central hole. The result lends support to the hypothesis that Sulfolobus chaperonin participates in ribosomal RNA maturation and ribosome assembly.

Interfacial properties of nonionic micellar aggregates as a function of temperature and concentration

Abstract By means of density, dielectric spectroscopy and sound velocity measurements we perform a systematic study on the polyoxyethylene C12E6 nonionic surfactant solutions as a function of temperature and concentration. Both density and sound velocity data, at about 34°C, coincide with the value obtained for pure water. Above this temperature the density is lower than the water density whereas below, it is greater, the opposite happens for the compressibility. Combining results from these different techniques we attempt a detailed description of the evolution of the micellar interfacial properties with temperature. It is well known that nonionic surfactant solutions dehydrate with growing temperature. Our results indicate that this process is associated with a continuous change in the polymer conformation and in the local density of the micellar interface.