The course integrates lectures (4 cfu) and lab activities (2 cfu). Topics: protein structure and different folds. Alpha proteins, alpha/beta proteins, beta proteins. Proteostasis. Folding and misfolding. Introduction to protein visualization software.
Enzymes. Thermodynamics and Michaelis-Menten Kinetics. Inhibition. Mechanisms of action of enzymes. Kinetics of prestationary state. Enzyme regulation. Mutagenesis and analysis of mutated enzymes. Scatchard plot. Crystallography in enzymology.
A. Fersht
STRUTTURA E MECCANISMI D’AZIONE DEGLI ENZIMI.
Zanichelli
Come funzionano le proteine
di Mike Williamson Bolognesi M. Zanichelli, 2013
Learning Objectives
Aim of this course is to provide students with the theoretical and practical fundamentals of the studies concerning the structure of proteins and enzymes. At the end of the course the student will be able to understand structure-function relationships, to analyse mechanisms of regulation and conformational flexibility of proteins, to predict and analyse structural and functional properties of proteins and enzymes. This will be achieved by integrating theoretical lectures with practical sessions at the bench.
Prerequisites
Knowledge of the fundamentals of biochemistry.
Teaching Methods
32 hours of lectures (4 cfu) and 24 hours of laboratory (2 cfu). The lab part consists in 12 hours of introductory lectures and 12 hours of practical work at the bench.
Further information
.
Type of Assessment
Oral examination + brief dissertation about the results obtained during the work in the lab
Course program
THEORY (4 cfu)
Introduction: secondary structure of proteins; alpha helices, 3.10 helices, pi helices. Beta strands, loops, beta-turns; polyproline helix. Supersecondary structure. Topological diagrams; helix-turn-helix motif; beta-hairpin; greek key; other motifs. Protein structure analysis software: introduction to pyMOL. Protein databank; Structure prediction. Protein domains.: biological functions and properties. Alpha helix proteins: inter-helix contacts and structure of alpha helix proteins; four helix bundle; globin fold; Alpha/beta protein: TIM barrel structure; Rossmann fold. Beta proteins: barrel proteins; greek key motif; jelly roll. Extremophile proteins: structural adaptations in thermophilic, hyperthermophilic, psychrophilic, piezophilic, halophilic proteins. Prrotein folding and misfolding; folding in vitro and in vivo. Misfolding; aggregation-prone conformational states. Proteostasis. Protein aggregation and protein misfolding diseases.
Properties of enzymes. Techniques to study protein structure: Coenzymes, cofactors and vitamins. The active site of enzymes. Classification of enzymes. The thermodynamics of enzymatic reactions. Spontaneity of a reaction. Activation energy (Ea) and Arrhenius equation. Relationship between Ea and enzymatic reaction rate. The enzyme kinetic. Michaelis-Menten equation. Main kinetic parameters: Km, Vmax and kcat. Effect of pH on an enzyme-catalyzed reactions. Dixon plot. Inhibitors and inhibition. Reversible and non reversible inhibition. Kinetic analysis of inhibition. Affinity labeling of catalytic residues. Examples of catalytic mechanisms. Pre-steady state kinetics. Identification of a kinetic intermediate. Regulation of enzyme activity. Control of synthesis and degradation. Covalent modifications. Proteolytic activation. Allosteric regulation of enzymes: principles and kinetic models. Study of the catalytic mechanism of an enzyme by mutagenesis. Structural and kinetic analysis of mutant enzymes. Dominant negative and identification of physiological substrates of the enzyme. Scatchard plot. Crystallography in enzymology
LAB (2 cfu)
Fluorescence spectra of floded and unfolded proteins; circular dichroism of proteins containing different types of structure. Dynamic light scattering of monomers and aggregates. Enzymatic activity assays; determination of the Km of an enzyme; determination of the Ki of an enzyme inhibitor.