Nicola Rosato

Fluorescence lifetime distributions in human superoxide dismutase. Effect of temperature and denaturation

The internal dynamics of human superoxide dismutase has been studied using time-resolved fluorescence. The fluorescence decay has been analyzed using continuous distribution of lifetime values. The effect of temperature and conformational state on the lifetime distribution has been investigated. The emission of the single tryptophan residue depends on the nature and dynamics of the protein matrix. Conformational changes have been induced by increased concentration of guanidinium hydrochloride. We found that both temperature and conformation strongly effect the width of the lifetime distribution.

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.

A stochastic model for the sigmoidal behaviour of cooperative biological systems

A stochastic model for cooperative transitions in biological systems based on a Markov chain is proposed. This model requires only two parameters, the mean probability, p, and the coupling capacity, Deltap, which measure the probability of forming a new weak bond depending on the number of similar bonds already formed and it is also responsible for the transition. In this paper we show how the model works for a large number of identical molecules and how it can be useful for studying the noise around the centre of the transition where, increasing the degree of cooperativity, i.e. the number n in the well-known Hill equation, the width of the noise increases along with its fractal dimension. A simple relationship between the degree of cooperativity and the parameter Deltap is proposed, suggesting that the cooperativity of real biological transitions is related to the coupling capacity Deltap of the present model.

Pressure dependence of protein dynamics investigated using elastic and quasielastic neutron scattering

We present here a study of the dynamics of two monomeric proteins, trypsin and lysozyme, by means of elastic and quasielastic neutron scattering under medium-to-high pressure conditions (1-1200 bar). The internal motions as probed from the average proton dynamics in the 100 ps timescale have a confined diffusive nature. On increasing the pressure up to 1200 bar the confinement volume is almost unaffected, while the fraction of protons involved is slightly decreased.

Dynamics of trypsin under pressure

Abstract The pressure dependence of the dynamics of a monomeric protein has been studied by quasielastic neutron scattering. The EISF shows a reduction in the volume sampled by the protons in going from 1 to 900 atm. A slight decrease of the quasielastic broadening with pressure is also observed.

A STOCHASTIC MODEL FOR THE COOPERATIVE RELAXATION OF PROTEINS, BASED ON A HIERARCHY OF INTERACTIONS BETWEEN AMINO ACIDIC RESIDUES

In this paper, a stochastic model for the cooperative relaxation of proteins, based on a hierarchic structure of the interactions between amino acids is proposed. It relies on the arbitrary splitting of interactions into two classes, strong and weak, and tests the preponderance of one class over the other. The presented model generalizes a first one valid for homogeneous interactions in the protein molecules previously studied by the authors. The time evolution of the system is studied as a function of five parameters, three of which are related to the cooperativity. Moreover, different approximations of the discrete system to a diffusion process, and to a Poisson process are considered, according to the magnitude of the parameters. A method for estimating the parameters from real data is proposed. Finally, numerical simulations and a comparison with the molecular dynamics of a real protein (Barnase) are reported.

Dynamic fluorescence in copper proteins Selected examples

SummaryThe fluorescence properties of three copper proteins, namely human superoxide dismutase,Pseudomonas aeruginosa azurin andThiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.

Azurins and Their Site-Directed Mutants

The detailed knowledge of azurin structure and the new possibil-ities offered by site-directed mutagenesis make it a convenient model for studies on the stability of small globular proteins. In particular the importance of a very stable hydrophobic core for maintaining the native, biologically active conformation appears evident. The peculiar location of the single tryptophan, just at heart of this core, has two important consequences. First, the spectroscopic features of this tryptophan are similar to those of indole in non-aqueous solutions and very low tem-peratures, even though, as demonstrated by the anisotropy decay, it has a considerable freedom of rotation. Second, it represents a very useful, built-in probe not only of the native-denatured transition, but also of subtler modifications of the structure which may preceed its collapse toward a disordered state.

Near-infrared spectroscopy of the human brain: effects of apnea and hypercapnia on the intensity and phase of backscattered light

The effects of hypercapnia on the intensity and phase of near infrared light back-scattered by the head were studied on eight healthy humans and two patients affected by monohemispheric lesions in the Middle Cerebral Artery (MCA) territory. A decrease in the light intensity and a variation of the phase were detected in all healthy subjects during apnea and hypercapnia. Only negligible changes were observed in the affected hemisphere of the patients. A concomitant study by Transcranial Doppler Sonography (TCD) showed an increase of the blood flow during hypercapnia both in normal hemispheres and, to a less extent, in the affected hemisphere of patients. This suggests that NIRS (Near Infrared Spectroscopy) is more sensitive to alterations of more cortical brain vascular system than TCD which is mainly testing MCA in the depth.

Effects on Intrinsic Fluorescence Induced by Active Site Modifications of Rhodanese

Rhodanese is a mitochondrial enzyme of unknown physiological function catalyzing “in vitro” the transport of zero valence sulfur from an appropriate donor like thiosulfate to a nucleophilic acceptor. The enzymic reactions occur via a double-displacement with the intermediacy of a sulfur-loaded form of enzyme where the zero valence sulphur is bound to an active site cysteine (CYS 247) in a persulfide linkage (Westley,1973).