Protein structure and dynamics

Research Lines: MD.P01.008.001 – Protein structure and dynamics
People: Patrizia Cioni, Edi Gabellieri, Andrea Mozzarelli, Samanta Raboni, Stefano Bettati, Luca Ronda, Serena Faggiano

The general objective is to investigate the relationship between structure / dynamics and function in proteins, mainly using tryptophan fluorescence and phosphorescence spectroscopies. The methodologies and instrumental devices developed in our laboratory allow the study of structural and dynamic properties of proteins in different experimental media and conditions such as: aqueous solutions, biological membranes, ice, dry state, micelles, silica gel, thin films, proteins adsorbed on solid support.
In addition, our instrumentation is used for high pressure studies, up to 700 MPa.

Spectroscopic approaches are used to reveal conformational changes induced by multiple factors such as:

  • binding (substrates, cofactors, inhibitors)
  • change in physical conditions (temperature, pressure, salinity, dehydration)
  • interaction with stabilizing or destabilizing agents
  • aggregation-dissociation of proteins.

 

Activities:

  • Development of a new drug for cancer therapy based on the enzyme methionine gamma lyase (MGL). In order to transport MGL to cancer cells and increase its bioavailability, the protein is conjugated to citrate-coated gold nanoparticles (synthesized in our laboratory) or PEG and the cytotoxicity of the AuNPs-MGL complex is evaluated on a panel of cancer cells . The physico-chemical properties of the complex (enzymatic activity, degree of bond, size distribution, surface area, quality and stability of conjugates, influence of gold nanoparticles on the structure of proteins, distribution and orientation of the MGL in the interfaces) are investigated also in collaboration with other structures.
  • Characterization of the structural determinants of stability and flexibility. In proteins function, thermodynamic stability, specificity and binding affinity are in many cases strictly dependent on structural fluctuations. The design of proteins with specific functions and stability may require the insertion of structural characteristics that allow dynamic excursions of the structure. For an effective protein design, a better understanding of the relationship between structure and flexibility is paramount. Using as a model azurin purified from Pseudomonas aeruginosa and some of its specifically constructed mutants, the effects of inserting cavities on flexibility and stability are investigated. By changing pressure and temperature, reversible denaturation experiments are carried out. The work allows to determine thermodynamic parameters such as ΔG, ΔV, ΔCp, Δα, Δβ contributing to the description of the denaturation process.
  • Denaturation of proteins at low temperature. The study of low temperature denaturation is fundamental to understand the forces that determine protein folding. A protein has been recently identified, yeast frataxin (Yfh1), whose cold denaturation occurs at experimentally accessible temperatures, close to 0 ° C and in physiological conditions, without the need to add denaturants. Experiments are conducted by varying both pressure and temperature with the aim of measuring the thermodynamic parameters of denaturation and helping to clarify the mechanism behind cold denaturation.
  • Study of intrinsically disordered proteins. By fluorescence measurements the responses of proNGF and NGF to pressure are characterized. The comparison between the two proteins allows to evaluate the contribution of the pro-peptide, intrinsically disordered protein, to the stability of the protein structure.

 

Keywords: fluorescence, phosphorescence, high pressure, proteins, stability, flexibility, folding, protein aggregation, cold denaturation, gold nanoparticles

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