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Minute, Fabrizio (2013) Entrapment of hemocyanin conformers as a tool for the definition of the structural model of cooperativity. [Tesi di dottorato]

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Abstract (inglese)

Allostery has been established as a fundamental mechanism of regulation in most biological processes (Changeux, 2012). The most studied allosteric protein is hemoglobin, that has long been considered a paradigm for the investigation of allostery and cooperativity in macromolecules (Cui & Karplus, 2008). A phenomenological characterization of the cooperative oxygen binding in hemoglobin has been given by the classic Monod, Wyman, Changeux model (MWC) (Monod et al., 1965), which described the cooperativity process as the allosteric transition between two different allosteric conformers: the high oxygen affinity R-state and the low oxygen affinity T-state.
In this work we wanted to characterize the highly cooperative oxygen binding process of the hemocyanin (in its dodecameric state) from the arthropod Carcinus aesturaii, which represents a more complex case study. To this aim the functional and structural properties of the different conformational states were investigated, through the encapsulation of the hemocyanin into a sol-gel matrix to avoid the allosteric transition between T- and R-state.
Firstly we characterized the structural and optical properties of the sol-gel matrix in order to find the better conditions for protein encapsulation. The effect of the pH, glycerol and aging time was assessed by collecting Small Angler X-Ray Scattering spectra of undoped sol-gel matrices at ELETTRA (Trieste, Italy). The fractal analysis of all the spectra did not evidence any significant effect of the three factors on the properties of the matrix, with the exception of the decrease of the radius of the silica particles that form the gel network in presence of glycerol. Moreover, the spectroscopic analysis of the undoped matrix revealed a good transparence of the matrix.
The key point of the work was to evaluate if the sol-gel matrix was a suitable way for the entrapment of hemocyanin in a single conformational state, through the analysis of the oxygen binding curves and the Hill-plots of the hemocyanin entrapped into the silica matrix (in presence and absence of oxygen). The results suggested that the sol-gel matrix can avoid the allosteric transitions, although the full entrapment of distinct conformers strongly depend on the pH. Oxygen binding experiments performed in presence of different concentrations of salt demonstrated that changes of the charge to charge interactions between protein and matrix may play an important role in conformational entrapment. The entrapment of different conformers was confirmed by spectroscopic analysis that revealed small but significant differences in the shape of the CD-spectra in the aromatic region and shifts of the peaks of fluorescence emission for the two conformers.
To verify if the oxygen equilibrium data were in agreement with the MWC model we compared the equilibrium constants of the curves obtained by the hemocyanin entrapped into the sol-gel with those predicted by the model, suggesting that the cooperativity in this hemocyanin occurs in a more complicated way. A three-state model was proposed.
The validation of the three-state model was performed through Small Angle Neutron Scattering (SANS) measurements at ILL neutron radiation facility (Grenoble, France), that allowed us to characterize the structure of the conformers both in the oxy- and deoxygenated state. The analysis of the SANS spectra seems to confirm the presence of three different conformers, with a different arrangement of the two hexamers.
In order to determine if these differences in the quaternary structure were correlated to changes in the active sties, we collected XAS spectra at ESRF (Grenoble, France), which shown differences between the different conformers, suggesting a different coordination geometry of the active sites in the deoxygenated form.
An investigation of the functional and structural effects of the positive allosteric effector lactate was also performed, by the analysis of oxygen binding curves and SANS spectra.
In conclusion, with this work it is defined a structural model of cooperativity for hemocyanin, with a functional and structural characterization of the different conformers. We also proposed a possible mechanism to describe how the quaternary conformational changes are linked to the different coordination geometry of the active site during the oxygen binding process.
This study method can be exported to other cooperative systems and the results also provide a solid start point for the development of an oxygen biosensor.

Abstract (italiano)

Allostery has been established as a fundamental mechanism of regulation in most biological processes (Changeux, 2012). The most studied allosteric protein is hemoglobin, that has long been considered a paradigm for the investigation of allostery and cooperativity in macromolecules (Cui & Karplus, 2008). A phenomenological characterization of the cooperative oxygen binding in hemoglobin has been given by the classic Monod, Wyman, Changeux model (MWC) (Monod et al., 1965), which described the cooperativity process as the allosteric transition between two different allosteric conformers: the high oxygen affinity R-state and the low oxygen affinity T-state.
In this work we wanted to characterize the highly cooperative oxygen binding process of the hemocyanin (in its dodecameric state) from the arthropod Carcinus aesturaii, which represents a more complex case study. To this aim the functional and structural properties of the different conformational states were investigated, through the encapsulation of the hemocyanin into a sol-gel matrix to avoid the allosteric transition between T- and R-state.
Firstly we characterized the structural and optical properties of the sol-gel matrix in order to find the better conditions for protein encapsulation. The effect of the pH, glycerol and aging time was assessed by collecting Small Angler X-Ray Scattering spectra of undoped sol-gel matrices at ELETTRA (Trieste, Italy). The fractal analysis of all the spectra did not evidence any significant effect of the three factors on the properties of the matrix, with the exception of the decrease of the radius of the silica particles that form the gel network in presence of glycerol. Moreover, the spectroscopic analysis of the undoped matrix revealed a good transparence of the matrix.
The key point of the work was to evaluate if the sol-gel matrix was a suitable way for the entrapment of hemocyanin in a single conformational state, through the analysis of the oxygen binding curves and the Hill-plots of the hemocyanin entrapped into the silica matrix (in presence and absence of oxygen). The results suggested that the sol-gel matrix can avoid the allosteric transitions, although the full entrapment of distinct conformers strongly depend on the pH. Oxygen binding experiments performed in presence of different concentrations of salt demonstrated that changes of the charge to charge interactions between protein and matrix may play an important role in conformational entrapment. The entrapment of different conformers was confirmed by spectroscopic analysis that revealed small but significant differences in the shape of the CD-spectra in the aromatic region and shifts of the peaks of fluorescence emission for the two conformers.
To verify if the oxygen equilibrium data were in agreement with the MWC model we compared the equilibrium constants of the curves obtained by the hemocyanin entrapped into the sol-gel with those predicted by the model, suggesting that the cooperativity in this hemocyanin occurs in a more complicated way. A three-state model was proposed.
The validation of the three-state model was performed through Small Angle Neutron Scattering (SANS) measurements at ILL neutron radiation facility (Grenoble, France), that allowed us to characterize the structure of the conformers both in the oxy- and deoxygenated state. The analysis of the SANS spectra seems to confirm the presence of three different conformers, with a different arrangement of the two hexamers.
In order to determine if these differences in the quaternary structure were correlated to changes in the active sties, we collected XAS spectra at ESRF (Grenoble, France), which shown differences between the different conformers, suggesting a different coordination geometry of the active sites in the deoxygenated form.
An investigation of the functional and structural effects of the positive allosteric effector lactate was also performed, by the analysis of oxygen binding curves and SANS spectra.
In conclusion, with this work it is defined a structural model of cooperativity for hemocyanin, with a functional and structural characterization of the different conformers. We also proposed a possible mechanism to describe how the quaternary conformational changes are linked to the different coordination geometry of the active site during the oxygen binding process.
This study method can be exported to other cooperative systems and the results also provide a solid start point for the development of an oxygen biosensor.

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Tipo di EPrint:Tesi di dottorato
Relatore:Beltramini, Mariano
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > BIOSCIENZE E BIOTECNOLOGIE > BIOTECNOLOGIE
Data di deposito della tesi:30 Gennaio 2013
Anno di Pubblicazione:30 Gennaio 2013
Parole chiave (italiano / inglese):hemocyanin cooperativity sol-gel allostery
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/09 Fisiologia
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:5828
Depositato il:16 Ott 2013 08:13
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