Biomedical subjects

S Cannistraro

Publications and source records attributed to S Cannistraro.

At least 19 recordsLinked to original sources

PubMed2006

Optimized biorecognition of cytochrome c 551 and azurin immobilized on thiol-terminated monolayers assembled on Au(111) substrates.

Molecular recognition between two redox partners, azurin and cytochrome c 551, is studied at the single-molecule level by means of atomic force spectroscopy, after optimizing azurin adsorption on gold via sulfhydryl-terminated alkanethiol spacers. Our experiments provide evidence of specific interaction between the two partners, thereby demonstrating that azurin preserves biorecognition capability when assembled on gold via these spacers. Additionally, the measured single-molecule kinetic reaction rate results are consistent with a likely transient nature of the complex. Interestingly, the immobilization strategy adopted here, which was previously demonstrated to favor electrical coupling between azurin (AZ) and the metal electrode, is also found to facilitate AZ interaction with the redox partner, if compared to the case of AZ directly adsorbed on bare gold. Our findings confirm the key role of a well-designed immobilization strategy, capable of optimizing both biorecognition capabilities and electrical coupling with the conductive substrate at the single-molecule level, as a starting point for advanced applications of redox proteins for ultrasensitive biosensing.

Azurin↗

PubMed2005

Single molecule recognition between cytochrome C 551 and gold-immobilized azurin by force spectroscopy.

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.

Adsorption↗

PubMed2004

Scanning tunneling spectroscopy investigation of self-assembled plastocyanin mutants onto gold substrates under controlled environment.

The study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol group (PCSH) or a disulfide bridge (PCSS) to covalently bind at gold surface. The I-V measurements were performed by positioning the STM tip on top of a single molecule and sweeping the bias potential between +/-1 V, under both ambient and controlled atmosphere. For PCSS, under ambient conditions, asymmetric I-V characteristics were obtained, which disappear under nitrogen atmosphere. PCSH, instead shows a symmetric I-V relation in air and under nitrogen environment. Here, as factors underlying this distinct electron conductive behaviour, a potential role for hydration water molecules and for copper redox levels are discussed.

Air↗

PubMed2002

A poplar plastocyanin mutant suitable for adsorption onto gold surface via disulfide bridge.

Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues Ile-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wild-type protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the full potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties.

Adsorption↗

PubMed2001

The 1.6 A resolution crystal structure of a mutant plastocyanin bearing a 21-25 engineered disulfide bridge.

Plastocyanin is an electron-transfer protein which has been largely used for biophysical studies as well as for protein-engineering experiments. A surface disulfide bridge has been engineered in poplar plastocyanin to allow protein chemisorption on gold substrates. The mutated plastocyanin crystal structure has been studied at 1.6 A resolution (R factor = 0.145, R(free) = 0.205) to characterize the effects of the engineered disulfide on the overall protein structure and on the Cu-coordination sphere in view of biophysical applications. The new orthorhombic crystal form isolated for the mutated plastocyanin displays two protein molecules per asymmetric unit.

Crystallization↗

PubMed2001

Molecular dynamics simulation and essential dynamics study of mutated plastocyanin: structural, dynamical and functional effects of a disulfide bridge insertion at the protein surface.

A molecular dynamics simulation (1.1 ns) at 300 K, of fully hydrated Ile21Cys, Glu25Cys plastocyanin mutant has been performed to investigate the structural, dynamical and functional effects of a disulfide bridge insertion at the surface of the protein. A detailed analysis of the root mean square fluctuations, H-bonding pattern and dynamical cross-correlation map has been performed. An essential dynamics method has also been applied as complementary analysis to identify concerted motions (essential modes), that could be relevant to the electron transfer function. The results have been compared with those previously obtained for wild-type plastocyanin and have revealed that the mutant shows a different pattern of H-bonds, with several interactions lost and a higher flexibility, especially around the electron transfer copper site. The analysis of dynamical cross-correlation map and of essential modes, has shown that the mutant performs different functional concerted motions, which might be related to the binding recognition with its electron transfer partners in comparison with the wild-type protein.

Algorithms↗

PubMed2001

Concerted motions in copper plastocyanin and azurin: an essential dynamics study.

Essential dynamics analysis of molecular dynamics simulation trajectories (1.1 ns) of two copper containing electron transfer proteins, plastocyanin and azurin, has been performed. The protein essential modes have been analysed in order to identify large concerted motions which could be relevant for the electron transfer function exerted by these proteins. The analysis, conducted for temporal windows of different lengths along the protein trajectories, shows a rapid convergence and indicates that for both the proteins the predominant internal motions occur in a subspace of only a few degrees of freedom. Moreover, it is found that for both the proteins the likely binding sites (i.e. the hydrophobic and negative patches) with the reaction partners move in a concerted fashion with a few structural regions far from the active site. Such results are discussed in connection with the possible involvement of large concerted motions in the recognition and binding interaction with physiological electron transfer partners.

Azurin↗

PubMed2001

Low-frequency vibrational modes in proteins: a neutron scattering investigation.

The low-frequency dynamics of plastocyanin, an electron transfer copper protein, has been investigated by incoherent neutron scattering at different temperatures. The contribution to the dynamic structure factor arising from H/D exchangeable and non-exchangeable protein protons has been evaluated by analyzing two differently exchanged protein samples. The dynamic structure factor of a hydrated plastocyanin sample with all the exchangeable hydrogens (about 150) replaced by deuterium exhibits an excess of vibrational modes, at about 3.5 meV, reminiscent of the boson peak found in other proteins and glassy systems. When only fast exchangeable hydrogens (about 50) are substituted by deuterium, the protein, besides the above-mentioned peak, shows an additional peak at about 1 meV. These vibrational peaks are discussed in connection with the topological disorder of the systems and the fluctuations of the intramolecular hydrogen bonds.

Biophysical Phenomena↗

PubMed2001

Potential-induced resonant tunneling through a redox metalloprotein investigated by electrochemical scanning probe microscopy.

The redox metalloprotein azurin self-chemisorbed onto Au(1 1 1) substrates has been investigated by electrochemically controlled scanning tunneling (STM) and scanning force/lateral force microscopy (SFM/LFM) and cyclic voltammetry (CV) in aqueous solution. The combined use of STM and SFM/LFM under electrochemical control in the negative side of the azurin redox midpoint (+116 mV vs. SCE) has delivered unique information on the nature of the STM images. While in STM the bright spots, believed to be associated with azurin molecules, are visible only for potential values higher than -125 mV, the concurrent electrochemical SFM results show adsorbed proteins over the whole potential range investigated (from -225 to +75 mV). Stepping the potential back and forth (between -25 and -125 mV) in STM imaging, it has been possible to make bright spots appearing and disappearing repeatedly, indicating that STM image formation arises possibly through resonant tunneling via the redox levels of azurin. These results represent the first clear evidence of potential-dependent tunneling in proteins adsorbed onto a conductive substrate.

Journal Article↗

PubMed2000

In situ Raman microspectroscopic identification and localization of carotenoids: approach to monitoring of UV-B irradiation stress on Antarctic fungus.

The in situ Raman microspectroscopic properties of an Antarctic fungus are investigated to assess the nature and the spatial localization of the main chromophores and to study their spectral changes under enhanced UV-B irradiation. The Raman spectroscopic features of spores in situ are consistent with those of carotenoid-like pigments. In particular, the Raman shifts seem to be related either to the frequency modes of long conjugated double-bond carotenoids or to protein bound beta-carotene. The spectroscopic analysis at different spore depths clearly shows the strongest Raman signal arises from cell wall and membrane structures. The intensity of such a signal shows a drastic reduction upon UV-B irradiation without any significant frequency change. The use of Raman microspectroscopy for nondestructively monitoring the UV-B effects on Arthrobotrys ferox spores is also discussed.

Antarctic Regions↗

PubMed2000

Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation.

We combined two-photon fluorescence microscopy and spectroscopy to provide functional images of UV-B (280-315 nm) induced stress on an Antarctic fungus. Two-photon excitation microscopy was used to characterize the distribution of autofluorescence inside the spore and the hyphae of the fungus. The imaging analysis clearly shows that the autofluorescence response of spores is higher than that of hyphae. The imaging analysis at different depths shows that, strikingly enough, the spore autofluorescence originates from the cell wall and membrane fluorophores. The spectroscopic results show moreover that the fluorescence spectra of spores are redshifted upon UV-B irradiation. Tentative identification of the chromophores involved in the autofluorescence response and their biological relevance are also discussed on the basis of a previous steady-state fluorescence spectroscopic study performed on both whole spore suspension and organic-soluble extracts.

Image Processing, Computer-Assisted↗

PubMed2000

Glasslike dynamical behavior of the plastocyanin hydration water.

The dynamical behavior of water around plastocyanin has been investigated in a wide temperature range by molecular dynamics simulation. The mean square displacements of water oxygen atoms show, at long times, a t(alpha) trend for all temperatures. Below 150 K, alpha is constant and equal to 1; at higher temperatures it drops to a value significantly smaller than 1, and thereafter decreases with increasing temperature. The occurrence of such an anomalous diffusion matches the onset of the dynamical transition observed in the protein. The intermediate scattering function of water is characterized, at high temperature, by a stretched exponential decay evolving, at low temperature, toward a two step relaxation behavior, which becomes more evident on increasing the exchanged wave vector q. Both the mean square displacements and the intermediate scattering functions show, beyond the ballistic regime, a plateau, which progressively extends for longer times as long as the temperature is lowered, such behavior reflecting trapping of water molecules within a cage formed by the nearest neighbors. At low temperature, a low frequency broad inelastic peak is observed in the dynamical structure factor of hydration water; such an excess of vibrational modes being reminiscent of the boson peak, characteristic of disordered, amorphous systems. All these features, which are typical of complex systems, can be traced back to the glassy character of the hydration water and suggest a dynamical coupling occurring at the macromolecule-solvent interface.

Computer Simulation↗

PubMed1999

Long-term molecular dynamics simulation of copper azurin: structure, dynamics and functionality.

A long-term molecular dynamics simulation (1.1 ns), at 300 K, of fully hydrated azurin has been performed to put into relationship the protein dynamics to functional properties with particular attention to those structural elements involved in the electron transfer process. A detailed analysis of the root mean square deviations and fluctuations and of the intraprotein H-bonding pattern has allowed us to demonstrate that a rigid arrangement of the beta-stranded protein skeleton is maintained during the simulation run, while a large mobility is registered in the solvent-exposed connecting regions (turns) and in the alpha-helix. Moreover, the structural elements, likely involved in the electron transfer path, show a stable H-bonding arrangement and low fluctuations. Analysis of the dynamical cross-correlation map has revealed the existence of correlated motions among residues connected by hydrogen bonds and of correlated and anti-correlated motions between regions which are supposed to be involved in the functional process, namely the hydrophobic patch and the regions close to the copper reaction center. The results are briefly discussed also in connection to the current through-bond tunneling model for the electron transfer process. Finally, a comparison with the structural and the dynamical behaviour of plastocyanin, whose structure and functional role are very similar to those of azurin, has been performed.

Journal Article↗

PubMed1999

Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results.

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the "boson peak" observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure.

Azurin↗

PubMed1999

Neutron scattering evidence of a boson peak in protein hydration water.

Measurement of the low temperature neutron excess of scattering of H2O-hydrated plastocyanin relative to D2O-hydrated protein allowed us to reveal the presence of an inelastic peak at about 3.5 meV. This excess of vibrational modes, elsewhere termed "boson peak," is due to the dynamical behavior of the water molecules belonging to the H2O-hydration shell surrounding the protein. The relevance of the boson peak to the dynamical coupling between the solvent and the protein, and hence to the protein functionality is addressed.

Biophysical Phenomena↗

PubMed1997

Long-term molecular dynamics simulation of copper plastocyanin in water.

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.

Journal Article↗

PubMed1997

An EPR investigation on the structural heterogeneity in copper azurin and plastocyanin.

The effects of cooling rate and of solvent properties on the active site heterogeneity of two copper proteins, azurin and plastocyanin, have been investigated at low temperature by electron paramagnetic resonance spectroscopy. The spectra of theses proteins have been analyzed, by an accurate computer simulation, in terms of a distribution of some relevant spin-Hamiltonian parameters. The results show that the structural heterogeneity of both proteins, quantified by the width of the distribution in the g and A tensors, is affected by both the freezing procedure and the solvent composition. In particular, the g distribution width is found to be reduced in the slow cooling regime; such a reduction appearing more significant when glycerol is added to the protein solutions. Despite of the similarity in the copper ion microenvironments of the two proteins, the effects are more pronounced in azurin. The results are discussed also in connection with the role played by the solvent and the rate of freezing in featuring the conformational substate landscape.

Journal Article↗

PubMed1997

Solvent effects on the distribution of conformational substates in native and azide reacted Cu, Zn superoxide dismutase. An EPR study.

Native and azide reacted Cu, Zn superoxide dismutase in aqueous and mixed water-glycerol solution have been investigated by EPR spectroscopy at low temperature. An accurate computer simulation, based on a well established theoretical model which has been reformulated for rhombic symmetry, has shown that the EPR spectrum of the copper ion in the native protein shows a significant g and A strain in the parallel region. The strain arises from a distribution of the ligand field strengths onto the metal ion and this could be traced back to the existence of a multiplicity of conformational states in the protein molecule. The strain is reduced in the presence of azide which is known to bind directly to the copper atom and to give rise to a more relaxed configuration corresponding to a square pyramidal geometry in which the apical ligand occupies an elongated position. In both samples, addition of glycerol further reduces the strain, indicating that the solvent is directly coupled to the protein matrix, thereby modulating the structural heterogeneity displayed by the protein molecule.

Azides↗