Rita Guzzi

A Spectroscopic and Calorimetric Investigation on the Thermal Stability of the Cys3Ala/Cys26Ala Azurin Mutant

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the proteins overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.

Intramembrane Polarity by Electron Spin Echo Spectroscopy of Labeled Lipids

The association of water (D(2)O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D(2)O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the (14)N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.

Early stage aggregation of human serum albumin in the presence of metal ions

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70°C by monitoring the optical density changes at 400nm on a 180min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu 2+ and Zn 2+ metal ions

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu2+ or Zn2+ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.

Intramembrane Water Associated with TOAC Spin-Labeled Alamethicin: Electron Spin-Echo Envelope Modulation by D2O

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D2O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D2O-ESEEM intensities decrease from TOAC(1) to TOAC(8) and TOAC(16) but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC(16) is located in the bilayer leaflet opposite to that of TOAC(1) and TOAC(8). Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D2O-ESEEM intensity is greatest for TOAC(8), indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.

Thermodynamics and kinetics of the thermal unfolding of plastocyanin

Abstract The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein.

Experimental model for the thermal denaturation of azurin: a kinetic study

Abstract The thermal denaturation of azurin in H 2 O, D 2 O and in ethanol-H 2 O mixtures has been investigated by electron spin resonance (ESR), optical absorption spectroscopy and differential scanning calorimetry (DSC). The OD 625 T variation observed at a scan rate of 0.7°C/min in H 2 O shows a cooperative OD transition between 78 and 82°C. In this step the intense charge-transfer band of azurin at 625 nm disappears. The ESR spectra recorded at − 153°C of the protein in the native state and after heating at 80 and 82°C indicate that both the symmetry and the copper ligands change with the thermal transition. The DSC measurements show that the thermal denaturation of azurin, which occurs at 84.4°C, is irreversible and kinetically controlled. This complex transition has been described as a multistep denaturation path and was analysed using a Lumry-Eyring type mechanism. The experimental C p.exc profile has been simulated and the calorimetric enthalpies related to the reversible and irreversible step, ΔH u and ΔH ag , respectively, are obtained. The kinetically controlled steps have been investigated by means of optical and DSC measurements at different scan rates and the apparent activation energy, E a , has been calculated. The denaturation of azurin in D 2 O and ethanol-H 2 O mixtures follows the same denaturation path as in H 2 O, although a shift of the OD 625 T and DSC profiles in evidenced. The temperature of the thermal transition and the E a values decrease in ethanol-H 2 O mixtures, but increase in D 2 O.

Conformational Heterogeneity and Spin-Labeled −SH Groups: Pulsed EPR of Na,K-ATPase

Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labeled on class I -SH groups in the presence of glycerol, or on class II -SH groups in the absence of glycerol. The class I-labeled preparations retain full enzymatic activity, whereas the class II-labeled preparations are at least partially inactivated. This provides an excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) can provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labeled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. 2H-ESEEM spectra of membranes dispersed in D2O reveal that class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or class II groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures (<150 K), but a more homogeneous conformational state at higher temperatures that is characterized by a single phase-memory T2M relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching an almost pure Lorentzian line shape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.

Evidence of reduced flexibility in disulfide bridge-depleted azurin: a molecular dynamics simulation study.

Two molecular dynamics simulations have been performed for 2 ns, at room temperature, on fully hydrated wild type and Cys3Ala/Cys26Ala double-mutant azurin, to investigate the role of the unique disulfide bridge on the structure and dynamics of the protein. The results show that the removal of the [bond]SS[bond] bond does not affect the structural features of the protein, whereas alterations of the dynamical properties are observed. The root mean square fluctuations of the atomic positions are, on average, considerably reduced in the azurin mutant with respect to the wild type form. The number of intramolecular hydrogen bonds between protein backbone atoms that are lost during the simulation, with respect to the starting configuration, are reduced in the absence of the disulfide bond. The analysis of the dynamical cross-correlation map, characterising the protein co-ordinated internal motions, demonstrates in the mutated azurin a significant decrease in anti-correlated displacements between protein residues, with the only exception occurring in the region of the mutation sites. The overall findings show a relevant reduction in flexibility as a consequence of the disulfide bridge depletion in azurin, suggesting that the [bond]SS[bond] bond is a structural element which significantly contributes to the dynamic properties of the native protein.

Chain dynamics in the low-temperature phases of lipid membranes by electron spin-echo spectroscopy

Spin-echo decays of spin-labelled phospholipids have been recorded to study the chain dynamics in the low-temperature phases of dipalmitoyl phosphatidylcholine membranes with and without 50 mol% cholesterol. The phase-memory relaxation time, T(2M), depends on the position of spin-labelling in the sn-2 chain, and on the presence of cholesterol. A biphasic temperature dependence of T(2M) is obtained over the range 150-270 K. Echo-detected field-swept absorption EPR spectra were recorded as a function of the echo delay time, tau. The echo-detected EPR lineshapes show a strong dependence on tau, revealing anisotropic phase relaxation arising from torsional chain motions. Cholesterol has a large effect on torsional oscillations about the chain long axis. Small-amplitude chain motions in the low-temperature phases may be important for cryopreservation of membranes.