Maurizio Leone

Abstract: At high temperature and low pH, the protein hormone insulin is highly prone to form amyloid fibrils, and for this reason it is widely used as a model system to study fibril formation mechanisms. In this work, we focused on insulin aggregation mechanisms occurring in HCl solutions (pH 1.6) at 60 degrees C. By means of in situ Thioflavin T (ThT) staining, the kinetics profiles were characterized as a function of the protein concentration, and two concurrent aggregation pathways were pointed out, being concentration dependent. In correspondence to these pathways, different morphologies of self-assembled protein molecules were detected by atomic force microscopy images also evidencing the presence of secondary nucleation processes as a peculiar mechanism for insulin fibrillation. Moreover, combining ThT fluorescence and light scattering, the early stages of the process were analyzed in the low concentration regime, pointing out a pronounced spatial heterogeneity in the formation of the first stable fibrils in solution and the onset of the secondary nucleation pathways.

Abstract: Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.

Abstract: Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies. In this work the effects of Cu(II) and Zn(II) ions on heat-induced structural modifications of bovine serum albumin (BSA) were studied, with the aim of delineating the role of these ions in the early stages of proteins aggregation kinetics. A joint application of different techniques was used. The aggregate growth was followed by dynamic light scattering measurements, whereas the conformational changes occurring in the protein structure were monitored by Raman and IR spectroscopy. Both in absence and in presence of metal ions, heating treatment gave rise to beta-structures to the detriment of alpha-helix conformation of BSA. The temperature of protein unfolding was not sensitively affected by the presence of Zn(II) or Cu(II) ions; on the contrary, only Zn(II) ions slightly promoted the heat-induced aggregation of the protein, since bigger aggregates were formed in their presence. The different efficacy of the Cu(II) and Zn(II) ions in promoting the BSA aggregation were highlighted by Raman measurements, assessing the role of His residues in metal binding. A distinct polypeptide folding of the two metal-BSA systems takes place, since the predominant mode of metal binding depends on metal. In particular, in Zn-BSA the metal coordination involves the imidazole N(tau) atom of His which can promote inter-molecular cross-linking.

Abstract: Protein cold-gelation has recently received particular attention for its relevance in bio and food technology. In this work, we report a study on bovine serum albumin cold-gelation induced by copper or zinc ions. Metal-induced cold-gelation of proteins requires two steps: during the first one, the heat treatment causes protein partial unfolding and aggregation; then, after cooling the solution to room temperature, gels are formed upon the addition of metal ions. The thermally induced behaviour has been mainly investigated through different techniques: Fourier transform infrared (FTIR) spectroscopy, circular dichroism, dynamic light scattering (DLS) and rheology. Data have shown that the aggregation process is mainly due to protein conformational changes--alpha-helices into beta-aggregates-forming small aggregated structures with a mean diameter of about 20 nm a few minutes after heating. After metal ion addition, the viscoelastic properties of the gels have been investigated by rheological measurements. The behaviour of the elastic and viscous moduli as a function of time is discussed in terms of ion concentration and type. Our results show that: (1) the elastic behaviour depends on ion concentration and (2) at a given ion concentration, gels obtained in the presence of zinc exhibit an elastic value larger than that observed in the Cu(2+) case. Data suggest that cold-gelation is the result of different mechanisms: the ion-mediated protein-protein interaction and the bridging effect due to the presence of divalent ions in solution.

Abstract: The fluorescent dye thioflavin T (ThT) is commonly used for in situ amyloid fibril detection. In this work, we focused on the spectroscopic properties and chemical stability of ThT in aqueous solution as a function of pH, temperature, and dye concentration. A reversible hydroxylation process occurs in alkaline solutions, which was characterized using a combination of UV-vis absorption spectroscopy, proton NMR, and density functional theory (DFT). On the basis of these studies, we propose a chemical structure for the hydroxylated form. Finally, by means of fluorescence spectroscopy, ThT hydroxylation effects on in situ amyloid detection have been investigated, providing new insights on the efficiency of the ThT assay for quantitative fibril evaluation at basic pH.

Abstract: At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.

Abstract: We report nanosecond time-resolved photoluminescence measurements on the isoelectronic series of oxygen deficient centers in amorphous silica related to silicon, germanium and tin atoms, which are responsible of fluorescence activities at approximately 4 eV under excitation at approximately 5 eV. The dependence of the first moment of their emission band on time and that of the radiative decay lifetime on emission energy are analyzed within a theoretical model able to describe the effects introduced by disorder on the optical properties of the defects. We obtain separate estimates of the homogeneous and inhomogeneous contributions to the measured emission line width, and we derive homogeneous spectroscopic features of the investigated point defects (Huang-Rhys factor, homogeneous width, oscillator strength, vibrational frequency). The results point to a picture in which an oxygen deficient center localized on a heavier atom features a higher degree of inhomogeneity due to stronger local distortion of the surrounding matrix. For Si, Ge, and Sn related defects, the parameter lambda, able to quantify inhomogeneity, is 65, 78, and 90%, respectively.

Abstract: We have recently shown that upon slight thermal destabilization the legume lectin Concanavalin A may undergo two different aggregation processes, leading, respectively, to amyloid fibrils at high pH and amorphous aggregates at low pH. Here we present an experimental study on the amyloid aggregation of Succinyl Concanavalin A, which is a dimeric active variant of Concanavalin. The results show that, as for the native protein, the fibrillation process appears to be favoured by alkaline pH, far from the isoelectric point of the protein. Moreover, it strongly depends on temperature and requires large conformational changes both at secondary and tertiary structure level. With respect to the native protein, the succinyl derivative forms amyloid fibrils in considerably longer times and with a minor exposure of hydrophobic regions. At physiological conditions, Concanavalin A still displays a sizeable tendency to form amyloid fibril, while the succinyl variant does not. A close correlation was observed between the progress of amyloid formation and a narrowing of the tryptophans fluorescence emission band, indicating a reduction of protein conformational heterogeneity in amyloid fibrils.

Abstract: In this work, we report a study of the effects of zinc and copper ions on the heat-induced aggregation of beta-lactoglobulin (BLG). Kinetics investigations on aggregates growth by light scattering measurements and on secondary structure changes by FTIR absorption measurements show the different role played by the two metals during the whole process. In particular, the presence of zinc in solution promotes the formation of aggregates of BLG at a lower temperature than copper. Then, at fixed temperature, formation of a large amount of aggregates, of large dimension, is observed for Zn-BLG in shorter time; on the contrary, the presence of copper in solution does not affect the aggregation process while the secondary structure changes and the formation of different stronger intermolecular H-bonds, which probably lead to build a network of bonds that takes towards gelation. Our studies show how time evolution of aggregation process of BLG is dramatically affected by the presence of metal ions in solution and structural protein modifications are induced by different divalent metal ions.

Abstract: We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.

Abstract: We report here a study on thermal aggregation of BSA at two different pH values selected to be close to the isoelectric point (pI) of this protein. Our aim is to better understand the several steps and mechanisms accompanying the aggregation process. For this purpose we have performed kinetics of integrated intensity emission of intrinsic and extrinsic dyes, tryptophans and ANS respectively, kinetics of Rayleigh scattering and of turbidity. The results confirm the important role played by conformational changes in the tertiary structure, especially in the exposure of internal hydrophobic regions that promote intermolecular interactions. We also confirm that the absence of electrostatic repulsion favours the disordered non-specific interactions between molecules and consequently affects the aggregation rate. Finally, the comparison between BSA and another relative protein, HSA, allows us to clarify the role of different domains involved in the aggregation process.

Abstract: Insulin, a 51-residue protein universally used in diabetes treatment, is known to produce amyloid fibrils at high temperature and acidic conditions. As for other amyloidogenic proteins, the mechanisms leading to nucleation and growth of insulin fibrils are still poorly understood. We here report a study of the fibrillation process for insulin confined in a suitable polymeric hydrogel, with the aim of ascertain the effects of a reduced protein mobility on the various phases of the process. The results indicate that, with respect to standard aqueous solutions, the fibrillation process is considerably slowed down at moderately high concentrations and entirely suppressed at low concentration. Moreover, the analysis of the initial stages of the fibrillation process in aqueous solutions revealed a large spatial heterogeneity, which is completely absent when the fibrillation is carried out in the hydrogel. We attribute this heterogeneity to the diffusion in solution of large amyloidal aggregates, which must be formed very fast compared to the average times for the whole sample. These findings are interpreted in the framework of recently suggested heterogeneous nucleation mechanisms. Moreover, they may be useful for the development of new insulin pharmaceutical formulations, more stable against adverse conditions.

Abstract: CdS nanopowder capped with sodium bis(2-ethylhexyl)sulfosuccinate was synthesized by using water-in-oil microemulsions. The CdS nanoparticles of about 5 nm obtained were embedded in polymethylmethacrylate matrix by a photocuring process. The transparent yellow solid compound was characterized by optical absorption and emission spectroscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The properties of this compound were compared with those of the nanopowder dispersed in heptane and in methylmethacrylate. The results obtained indicate that the nanoparticles are homogeneously dispersed in the matrix and do not change in size during the embedding process. Even if the surface slightly changes its luminescence properties, as a consequence of the different new chemical environment, the final product seems to be suitable for practical applications.

Abstract: In this work the temperature dependence of the Soret band line shape in carbon-monoxy myoglobin is re-analyzed by using both the full correlator approach in the time domain and the frequency domain approach. The new analyses exploit the full density of vibrational states of carbon-monoxy myoglobin available from normal modes analysis, and avoid the artificial division of the entire set of vibrational modes coupled to the Soret transition into "high-frequency" and "low-frequency" subsets; the frequency domain analysis, however, makes use of the so-called short-times approximation, while the time domain one avoids it. Time domain and frequency domain analyses give very similar results, thus supporting the applicability of the short-times approximation to the analysis of hemeprotein spectra; in particular, they clearly indicate the presence of spectral heterogeneity in the Soret band of carbon-monoxy myoglobin. The analyses also show that a temperature dependence of the Gaussian width parameter steeper than the hyperbolic cotangent law predicted by the Einstein harmonic oscillator and/or a temperature dependence of inhomogeneous broadening are not sufficient to obtain quantitative information on the magnitude of an-harmonic contributions to the iron-heme plane motion. However, the dependence of the previous two quantities may be used to obtain semiquantitative information on the overall coupling of the Soret transition to the low-frequency modes and therefore on the dynamic properties of the heme pocket in different states of the protein.

Abstract: To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein; disordered structures add as the pD decreases. Moreover, beta-aggregates seem to contribute only to oligomers formation, as showed by the good correlation between kinetics of scattering intensity and IR absorption intensity. These results indicate for BSA a general mechanism of aggregation composed by partial unfolding of the tertiary structure and by the decrease of alpha-helix and random coil contents in favor of beta-sheet aggregates. This mechanism strictly depends on pD and gives rise to almost two distinct types of macromolecular aggregates.

Abstract: Heme pocket dynamics of human carbonmonoxy hemoglobin (HbCO) is studied by Fourier transform infrared spectroscopy. The CO stretching band at various temperatures in the interval 300-10 K is analyzed in terms of three taxonomic A substates; however, in HbCO the band attributed to the A(1) taxonomic substate accounts for approximately 90% of the total intensity in the pH range 8.8-4.5. Two different regimes as a function of temperature are observed: below 160 K, the peak frequency and the bandwidth of the A(1) band have constant values whereas, above this temperature, a linear temperature dependence is observed, suggesting the occurrence of transitions between statistical substates within the A(1) taxonomic substate in this protein. The relationship between the heme pocket dynamics (as monitored by the thermal behavior of the CO stretching band), the overall dynamic properties of the protein matrix (as monitored by the thermal behavior of Amide II and Amide I' bands) and the glass transition of the solvent (as monitored by the thermal behavior of the bending band of water) is also investigated. From this analysis, we derive the picture of a very soft heme pocket of hemoglobin characterized by rather large anharmonic terms and strongly coupled to the dynamic properties of the solvent.

Abstract: We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the two tryptophans located in two different domains, in way to study conformational changes in the surrounding of these residues. We also follow emission spectra of Fluorescein-5-Maleimide dye bound to the single free cysteine of BSA. Complementary information on the extent of aggregation and on the structural changes is obtained by Rayleigh scattering and circular dichroism measurements. These data contribute to clarify the connection between conformational changes at tertiary and secondary structure level during the aggregation and how the different domains are involved. We also discuss the relevant role played by cysteine 34 in the aggregation pathways.

Abstract: We report the Soret absorption band, down to cryogenic temperature, of native and molten-globule-like state of horse heart cytochrome c. The band profile is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Franck-Condon approximation. From the temperature dependence of the Gaussian broadening and of the peak position, we obtain information on the 'bath' of low frequency harmonic motions of the heme group within the heme pocket. The reported data indicate that, compared to the native state, the less rigid tertiary structure of the molten globule is reflected in a higher flexibility of the heme pocket and in greater conformational disorder, allowing the transduction of large-amplitude motion of the protein to the dynamics of the heme pocket.

Abstract: The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.

Abstract: We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.

Abstract: Binding of azide to the native and arginine-modified bovine Cu,Zn superoxide dismutase in the oxidized and reduced form and to the copper-free derivative has been investigated by Fourier transform infrared spectroscopy. The antisymmetric stretching band of the azide is shifted to higher energy upon coordination to the copper atom of the oxidized form of the native enzyme. Similar spectral changes occur upon interaction of the anion with the Cu-diethylenetriamine model compound. On the other hand, interaction of azide with the native reduced form of the enzyme results in a band shift toward lower energy with respect to the free anion band. The same shift is observed after reaction of the azide with free lysine or arginine but not when it is reacted with other amino acid residues. The antisymmetric band of the azide is not perturbed by addition of the reduced arginine-modified enzyme; it is likely shifted toward higher energy upon addition of oxidized arginine-modified enzyme while it is again shifted toward lower energy in the presence of the copper-free derivative of the unmodified enzyme. It is concluded that azide does not directly coordinate to the copper in the reduced form of Cu,Zn superoxide dismutase but it remains in the active-site pocket in electrostatic interaction with the guanidinium group of Arg141, which is an invariant residue in this class of enzymes.

Abstract: Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Abstract: The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val --> Thr mutation has a larger conformational effect on the beta-subunits than on the alpha-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the alpha-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271-26278). These effects are attributed to a different electrostatic interaction between Ogamma of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Ogamma in the beta-subunit heme pocket.

Abstract: The dynamic and functional properties of mutant deoxyhemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified alpha chain and beta chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the alpha chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the beta chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified alpha chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the alpha chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified beta chains is not observed by x-ray crystallography.

Abstract:

Abstract: A normal-mode analysis of carbon monoxymyoglobin (MbCO) and deoxymyoglobin (Mb) with 170 water molecules is performed for (54)Fe and (57)Fe. A projection is defined that extracts iron out-of-plane vibrational modes and is used to calculate spectra that can be compared with those from resonance Raman scattering. The calculated spectra and the isotopic shift (57)Fe versus (54)Fe agree with the experimental data. At low temperatures the average mean square fluctuations (MSFs) of the protein backbone atoms agree with molecular dynamics simulation. Below 180 K the MSFs of the heme iron agree with the data from Mossbauer spectroscopy. The MSFs of the iron atom relative to the heme are an order of magnitude smaller than the total MSFs of the iron atom. They agree with the data from optical absorption spectroscopy. Thus the MSFs of the iron atom as measured by Mossbauer spectroscopy can be used to probe the overall motion of the heme within the protein matrix, whereas the Gaussian thermal line broadening of the Soret band and the resonance Raman bands can be used to detect local intramolecular iron-porphyrin motions.

Abstract: We report the Soret absorption spectra (500-350 nm) of the cyanomet derivatives of human hemoglobin and horse myoglobin, in the temperature range 300-20 K and in two different solvents (65% v/v glycerol-water or 65% v/v ethylene glycol-water). In order to obtain information on stereodynamic properties of active site of the two hemeproteins, we perform an analysis of the band profiles within the framework of electron-vibrations coupling. This approach enables us to single out the various contributions to the spectral bandwidth, such as those arising from non-radiative decay of the excited electronic state (homogeneous broadening) and from the coupling of the electronic transition i) with high frequency modes (that determines the vibronic structure of the band) and ii) with a "bath" of low frequency modes (that is responsible for the temperature dependence of the experimental spectra). We discuss the relevant parameters and their temperature dependence and compare them with the ones already reported for other derivatives of the same hemeproteins in the same solvents. In particular, non-harmonic contributions to soft modes are found, for cyanomet derivatives, to be larger than those observed for liganded carbonmonoxy but smaller than those observed for unliganded deoxy derivatives. The reported data enable us to obtain information on the dependence of stereodynamic properties of the heme pocket upon iron oxidation state, dimensions of the exogenous ligand and composition of the external matrix.

Abstract: The near-infrared charge transfer band at 760 nm (band III) has been investigated in deoxy and photodissociated dimeric Scapharca hemoglobin. At 300 K, the 10-ns spectrum of the carbonmonoxy derivative photoproduct is shifted by about 6 nm toward longer wavelengths with respect to the deoxy spectrum, both in buffer and in glycerol/buffer solutions. Moreover, the band III peak occurs at about the same wavelength at 300 K and at 10 K for the 10-ns photodissociated derivative, whereas in the deoxy derivative large changes in peak position and linewidth are observed as a function of temperature. These findings suggest that in dimeric Scapharca hemoglobin the photoproduct has not relaxed after 10 ns. The complete time dependence of the relaxation process has been studied both in buffer and in glycerol/buffer solutions at room temperature. The relaxation from the photoproduct to the deoxy species occurs on a microsecond time scale, in line with recent optical absorption and resonance Raman measurements.

Abstract: In this short review we show how suitable analysis of the temperature dependence of the optical absorption spectra of metalloproteins can give insight into their stereodynamic properties in the region of the chromophore. To this end, the theory of coupling between an intense allowed electronic transition of a chromophore and Franck-Condon active vibrations of the nearby atoms is applied to the Soret band of hemeproteins to obtain an analytical expression suitable for fitting the spectral profile at various temperatures. The reported approach enables one to separate the various contributions to the overall bandwidth together with the parameters that characterize the vibrational coupling. The thermal behavior of these quantities gives information on the dynamic properties of the active site and on their dependence upon protein structure and ligation state. The Soret band of hemeproteins appears to be coupled to high frequency vibrational modes of the heme group (as already shown by resonance Raman spectroscopy) and to a "bath" of low frequency modes most likely deriving from the bulk of the protein. For the deoxy derivatives inhomogeneous broadening arising from conformational heterogeneity appears to contribute substantially to the linewidth. The data indicate the onset, at temperatures near 180 K, of large scale anharmonic motions that can be attributed to jumping among different conformational substates of the protein.

Abstract: The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectivity replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N3(-)-Cu(II), Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(II) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm-1; the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Addition of N3- to the Cu(II),Co(II) SOD introduces slight changes in all the Co(II) visible bands, indicating the occurrence of minor perturbations of the structural cobalt site upon anion binding to the catalytic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(II) enzymes indicates that the His61 imidazolate bridge is released from the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the derivatives. The cobalt site is characterized by a harmonic dynamics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Polticelli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degree of local microheterogeneity at the cobalt site is smaller than that observed for the copper site and increases in the order N3(-)-Cu(II),Co(II) approximately Cu(II),Co(II) < Cu(I),Co(II) < E,Co(II) indicating a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic behavior with respect to the catalytic, solvent-accessible, copper site is discussed.

Abstract: The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some beta-chain mutant hemoglobins is studied in the temperature range 300-10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the over-all bandwidth are singled out with this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wild-type and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme-CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the "polar isosteric" beta Val-67(E11)-->Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue beta Lys-144(Hc1) at the C-terminal and residue beta Cys-112(G14) at the alpha 1 beta 1 interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the beta-N-terminal and at the alpha 1 beta 2 interface have no effect on the dynamic properties of the same heme pocket.

Abstract: The optical absorption spectra of native and N(3-)-reacted Cu,Zn superoxide dismutase (SOD) has been studied in the temperature range 300-10 K. The broad d-d bands observed in the room temperature spectrum, centered at 14,700 cm-1 (native enzyme) and at 15,550 cm-1 (N(3-)-reacted enzyme), are clearly split at low temperature into two bands each, centered at 12,835 and 14,844 cm-1 and at 14,418 and 16,300 cm-1, respectively. The thermal behavior of the 23,720 cm-1 band present in the spectrum of the native enzyme indicates that this band belongs to the His61-->Cu(II) ligand to metal charge transfer transition. Analysis of the zeroth, first, and second moments of the various bands as a function of temperature allowed us to obtain useful information on the stereodynamic properties of the metal site in SOD. In particular for the native protein, it was possible to infer a variation in the metal ligand relative position that occurs as the temperature is lowered and that likely involves all of the ligands except His61. On the other hand, the site is stabilized upon N3- binding, and in this case a variation in the metal ligand position is observed only at the level of the bound anion. The possible relation of these properties to the catalytic mechanism of the enzyme is discussed.

Abstract: The study of the thermal evolution of the Soret band in heme proteins has proved to be a useful tool to understand their stereodynamic properties; moreover, it enables one to relate protein matrix fluctuations and functional behavior when carried out in combination with kinetic experiments on carbon monoxide rebinding after flash photolysis. In this work, we report the thermal evolution of the Soret band of deoxy, carbonmonoxy, and nitric oxide derivatives of the cooperative homodimeric Scapharca inaequivalvis hemoglobin in the temperature range 10-300 K and the carbon monoxide rebinding kinetics after flash photolysis in the temperature range 60-200 K. The two sets of results indicate that Scapharca hemoglobin has a very rigid protein structure compared with other hemeproteins. This feature is brought out i) by the absence of nonharmonic contributions to the soft modes coupled to the Soret band in the liganded derivatives, and ii) by the almost "in plane" position of the iron atom in the photoproduct obtained approximately 10(-8) s after dissociating the bound carbon monoxide molecule at 15 K.

Abstract: We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300-20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset of substrate interconversions in these latter systems can be reflected in motions of the iron atom with respect to the porphyrin plane.

Abstract: In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.

Abstract: In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300-20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0-0 electronic transition frequencies. The reported analysis enables us to isolate the various contributions to the overall bandwidth, and their temperature dependence points out the relevance of low frequency vibrations and of large scale anharmonic motions starting at temperatures higher than 170 K. Information on the mean iron-heme plane distance and on its temperature dependence, as well as on the heme pocket conformational disorder, is also obtained.

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Abstract: In this work we study the temperature dependence of the Soret band lineshape of the carbonmonoxy derivatives of sperm whale myoglobin, human hemoglobin, and its isolated alpha and beta subunits. To fit the observed spectral profile we use an analytic expression derived for a system whereby a single electronic transition is coupled to Franck-Condon active vibrational modes, within the adiabatic and harmonic approximation. The vibronic structure of the spectra arises from the coupling with high frequency modes; these modes contribute to the total line shape through a series of Lorentzians with peak positions at vibrational overtones and half width related to the time constant of the population decay of the excited electronic state (homogeneous broadening); moreover, the coupling with low frequency modes broadens each Lorentzian to a Voigtian. Inhomogeneous broadening is modeled as a gaussian distribution of the 0-0 transition frequencies and is therefore added as a constant term to the previous gaussian width. This spectral deconvolution enables us to investigate the different contributions to line broadening and the parameters that characterize the vibrational coupling, as well as their dependence upon protein and solvent composition. The investigation is carried out as a function of temperature in the range 20-300 K; relevant information is obtained by comparing experimental results with theoretical predictions. This work supports a description of the investigated proteins as heterogeneous systems, whose heterogeneity depends on the particular protein and on the composition of the external matrix. The delocalized pi electron cloud of the porphyrin ring is coupled not only to the high frequency vibrational modes of the active site but also to a "bath" of lower frequency modes that involve the entire protein; moreover at suitable temperatures (approximately 200 K), anharmonic motions, which are an obvious prerequisite for the jumping among different conformational substates, become evident.

Abstract: We report the near infrared absorption spectra of cyanomethemoglobin and cyanometmyoglobin in two different solvents (deuterated solutions containing 65% v/v glycerol(OD)3 or 65% v/v ethylene glycol(OD)2). At 25 K the spectra show a clearly resolved fine structure that can be accounted for by considering a strong coupling of the porphyrin-to-iron charge transfer transitions with a single vibrational mode at 365 cm-1. The coupling constants depend on both the specific electronic transition and the protein surrounding the chromophore, indicating once more the specificity of heme globin interactions.

Abstract: We report the low temperature carbon monoxide recombination kinetics after photolysis and the temperature dependence of the visible absorption spectra of the isolated alpha SH-CO and beta SH-CO subunits from human hemoglobin A in ethylene glycol/water and in glycerol/water mixtures. Kinetic measurements on sperm whale (Physeter catodon) myoglobin and previously published optical spectroscopy data on the latter protein and on human hemoglobin A, in both solvents, (Cordone, L., A. Cupane, M. Leone, E. Vitrano, and D. Bulone. 1988. J. Mol. Biol. 199:312-218) are taken as reference. Low temperature flash photolysis data are analyzed within the multiple substates model proposed by Frauenfelder and co-workers (Austin, R. H., K. W. Beeson, L. Eisenstein, H. Frauenfelder, and I. C. Gunsalus. 1975. Biochemistry. 14:5355-5373). Within this model a distribution of activation enthalpies for ligand binding accounts for the structural heterogeneity of the protein, while the preexponential factor, containing also the entropic contribution to the free energy of the process, is considered to be constant for all conformational substates. Optical spectra are deconvoluted in gaussian components and the temperature dependence of the moments of the resulting bands is analyzed, within the harmonic Frank-Condon approximation, to obtain information on the stereodynamic properties of the heme pocket. The kinetic and spectral parameters thus obtained are found to be protein dependent also with respect to their sensitivity to changes in the composition of the external medium. A close correlation between the kinetic and spectral features is observed for the proteins examined under all experimental conditions studied. The results reported are discussed in terms of differences in the heme pocket structure and in the conformational heterogeneity among the various proteins, as related to their different capability to accommodate constraints imposed by the external medium.

Abstract: We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.

Abstract: We have measured the optical absorption spectra of azurin and stellacyanin in the wavelength range 1100-350 nm and in the temperature interval 290-20 K. Samples are protein aqueous solutions containing 65% (v/v) glycerol or ethylene glycol as cryoprotectants and remain homogeneous and transparent throughout the whole temperature range investigated. Spectra are deconvoluted into Gaussian components and the temperature dependence of the zeroth, first and second moments of the various bands is analyzed, within the harmonic Franck-Condon approximation, to obtain information on the stereodynamic properties of the active sites of these proteins. Sizable differences of the integrated intensities of all the bands with temperature are observed and are attributed to variations of the metal-ligand relative positions (i.e., deformations of the active site) that occur as the temperature is lowered. The mean effective frequency of the nuclear vibrations coupled to all the observed bands is about 150 cm(-1) for both proteins in both solvents used; this indicates that the electronic transitions from which the optical spectrum originates are substantially coupled with low-frequency vibrational modes, likely ligand-metal-ligand deformations. The relevance of the stereodynamic properties of azurin and stellacyanin, investigated in this work, to their functional behavior is also suggested.

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Abstract: The visible absorption spectra of carbonmonoxymyoglobin in the temperature range 300 to 20 K are reported and compared with the analogous spectra of carbonomonoxyhaemoglobin. The temperature dependence of the zeroth, first and second moment of the observed bands is analysed to obtain information on the local dynamics in the proximity of the haem. Contrary to haemoglobin, the first moment of the observed bands in myoglobin is markedly affected by the solvent composition and its value saturates at temperatures at which the solvent undergoes the glass transition. These data indicate that solvent properties influence the haem pocket stereodynamics in myoglobin; moreover, the different behaviour between myoglobin and haemoglobin suggests that the process should involve the surfaces that are buried in the haemoglobin tetramer and exposed to the solvent in myoglobin, and/or the different protein compressibility.

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Abstract: We have studied the optical absorption spectra of human deoxy- and oxyhemoglobin in the temperature range 300-20 K and in the wavelength range 350-1350 nm. By lowering the temperature, a narrowing and a shift of all bands were observed together with a sizeable increase of the integrated intensities of the charge-transfer bands of deoxyhemoglobin. At all temperatures the spectra are in full agreement with the band assignment previously suggested in the literature and no new relevant bands have been detected for both deoxy- and oxyhemoglobin. Analysis of the first and second moment of the bands, within the framework of the harmonic Franck-Condon approximation, gave information on the dynamic properties of the heme in the heme pocket.

Abstract: Thermodynamic interpretation of experiments with isotopically perturbed solvent supports the view that solvent stereodynamics is directly relevant to thermodynamic stability of biomolecules. According with the current understanding of the structure of the aqueous solvent, in any stereodynamic configuration of the latter, connectivity pathways are identifiable for their topologic and order properties. Perturbing the solvent by isotopic substitution or, e.g., by addition of co-solvents, can therefore be viewed as reinforcing or otherwise perturbing these topologic structures. This microscopic model readily visualizes thermodynamic interpretation. In conclusion, the topologic stereodynamic structures of connectivity pathways in the solvent, as modified by interaction with solutes, acquire a specific thermodynamic and biological significance, and the problem of thermodynamic and functional stability of biomolecules is seen in its full pertinent phase space.