69348 - Chemical and Molecular Methods for the Study of Proteins

Learning outcomes

At the end of the course, the student has acquired deep knowledge on some largely used methods to identify, produce and purify proteins. Moreover, he knows the physical bases and applications of some calorimetric, spectroscopic and light scattering techniques, aimed to characterize the conformation and the oligomeric state of proteins in solution, their conformational changes, the interactions occurring upon functioning. In particular, the student is able to: - propose the experimental strategies for protein isolation in their native state; - comprehend techniques that analyze the secondary and tertiary structure and conformational changes; - comprehend techniques to define the hydrodynamic properties of proteins in solution, such as molar mass and oligomeric state; - comprehend calorimetry to define the protein conformational state and protein-protein or protein-ligand interactions; - utilize some computer tools to analyze experimental data.

Course contents

Production of recombinant proteins. Expression of proteins in bacteria. Expression vectors and fusion proteins. Codon usage and genetic code. Increasing the protein yield and stability. Expression of protein in eukaryotic cells: insects, yeast, mammals, plants, cell-free.

Protein purification. Three phases of protein purification. Separation techniques. Salting in/out. Liquid chromatography. Affinity chromatography, ionic exchange, hydrophobic interaction, size exlusion. Designing a purification protocol. Testing protein purity.

Structural stability of proteins. Factors influencing protein stability in solution. Evaluating the denaturation temperature. Secondary and tertiary structure. Circular dichroism and spectra interpretation. Thermo-shift assay. Differential scanning calorimetry. Conformational ensemble. Structure-function paradigm and intrinsically disordered proteins.

Molecular interactions. Studying protein-protein, protein-ligand and protein-DNA interactions. Theoretical basis of Isothermal titration calorimetry. Calorimetry to study enzyme kinetics. Quaternary structure and hydrodinamic properties using static and dynamic light scattering. How to interpret the experimental data, using specific software.

Readings/Bibliography

There is not need to buy specific texts. Students will be given scientific articles and lesson material through Dropbox.

Teaching methods

Lectures (32 hours) will be in traditional classes. Four hours of practical lessons of data analysis, with each student working on individual computers, will be included, in order to illustrate specific software to analyze calorimetric and spectroscopic data. This part of the course will be carried out in the Bioinformatic Lab (Navile).

Assessment methods

At the end of the course, each student will be tested for having reached the didactic objectives and to verify that he had acquired the theoretical and practical knowledge on methods for i) producing recombinant proteins, ii) studying their biochemical-structural and hydrodynamic properties, iii) evaluating conformational changes following structural perturbation and iv) defining biomolecular interactions. Each student will choose an argument, among the ones described in the course, on which he will do a bibliographic research, the result of which will be illustrated during the oral exam. In addition, the student will be required to answer one or two questions on the main subjects of the course. The final evaluation will be an average between the research exposition and the ability of the student to answer to the question proposed.

Teaching tools

Articles and slides will be shared with students through Internet.

Office hours

See the website of Barbara Zambelli