Learning objectives
At the end of the course the student is expected to be able to:
- Interpret the structural and functional aspects of coordination systems of relevance in biology on the basis of the fundamental concepts of complex chemistry.
- Exemplify types of bioinorganic systems and classify them using the classes of systems studied
- Compare two different bioinorganic systems (eg. two different metalloproteins) and analyze similarities and differences
- Check the correctness of the experimental design of a laboratory experiment (of the type of those carried out during the course) on the basis of the starting data and of the objectives of the experiment
- To plan a laboratory experiment (of the type of those carried out during the course) given the objective of the same
Prerequisites
Basis of General Chemistry, Organic Chemistry, and Biochemistry.
Course unit content
The course deals with the role of metals in biological and biomolecular systems, with particular reference to substrate binding and catalytic activity of metalloproteins, interaction of metal ions with relevant peptides in neurodegenerative diseases, and role of metal centers in electron transport. During the course will be also presented the strategies of protein design (redesign and de novo design) with particular reference to metalloproteins.
Part of the course (2 CFU) will be carried out in the form of lab activities (mainly studies of metalloproteins) and the use of software for the study of bioinorganic systems.
Topics
Introduction to the course
Coordination compounds (or complexes) - Inroduction
Coordination compounds - Biological relevance
Localization and transport of metals in biological systems
Electron transfer processes
Metalloproteins and metalloenzymes
Metal ions in neurodegenerative disorders
Protein Design and Metalloprotein Design
Introduction to laboratory activities
Lab activity 1. Spectrophotometric and CD characterization of serum albumin (SA) and of its adducts with Cu (II) and Cu (I) - Reduction of Cu (II) / SA adduct to Cu (I) / SA - Metallochromic indicators .
Lab activity 2. Determination of the distribution of metal ions in a pool of bioligands in solution using the software HySS.
Exercise 3. Study of the binding of oxygen and carbon monoxide to myoglobin with UV-Vis absorption spectrophotometry.
Exercise 4. Determination of Michaelis and Menten parameters for the oxidation reaction of phenylenediamine catalyzed by horseradish peroxidase - Cysteine as inhibitor - NMR characterization of substrate and inhibitor.
Full programme
Introduction to the course(1 h)
Coordination compounds (or complexes) - Inroduction (5 h)
- Metal ions of the d and f series. Ligands. Coordination number and geometry. Equilibria of formation of the complexes and their stability. Ligand Field Theory. Spectroscopic and Magnetic Properties. Hard-Soft theory. Chelate effect.
Coordination compounds - Biological relevance (2 h)
- Introduction to the biological relevance of coordination compounds. Amino acids as ligands. Acid-base and and complexation properties of amino acid residues. Role of the backbone. Nucleobases as ligands.
- Theory of Lab Activity 1.
Localization and transport of metals in biological systems (2 h)
- Biological ligands: from small molecules to peptides. Role of stability of the complexes. Kinetic aspects.
- Chemical buffers in biotechnology and their interaction with metal ions. Artificial chelating agents.
- Case study: "Affinity gradients drive copper to cellular destinations".
- Theory of Lab Activity 2.
Electron transfer processes (3 h)
Metal ions with more than one oxidation state: chemical properties and implications in the formation of complexes. Hard Soft Properties in relation to redox states. Electron transfer reactions in the complexes: outer sphere and inner sphere processes. Brief introduction to the Marcus theory.
- Metal ions with more than one redox state: relevance in biology. Oxidants and reducing agents in biology. Oxidation and reduction products. Electron flows in proteins
Metalloproteins and metalloenzymes (4 h)
- Introduction to the role of metal ions in proteins. Structural, transport and catalytic centers.
- Two classes of metalloproteins: copper proteins and iron - heme proteins. Structural and functional aspects.
- Theory of Lab Activity 3.
- Theory of Lab Activity 4
Metal ions in neurodegenerative disorders (ND) (3 h)
- Relevant biomolecules in Alzheimer, Parkinson, Creutzfeldt-Jacob neurodegenerative diseases: beta amyloid, alpha synuclein, tau, prion, and their adducts with metal ions. Aggregation. Oxidative stress. Chelation therapy for metals: the case of Cu (II) in Alzheimer's disease.
Protein Design and Metalloprotein Design (8 h)
- Principles and purpose of protein design. Protein Redesign. De novo design. Design of the first and second coordination sphere of metal sites. Positive and negative design. Rational and combinatorial approach. In silico design.
- Protein Redesign: Redesign of metal coordination sites; redesign of prosthetic groups; directed evolution.
- De novo design: alpha-helix constructs; beta sheets constructs; mixed constructs.
- Alpha-helix constructs. Helical coiled coils: parallel and antiparallel coiled coils. Homomeric and heteromeric constructs. Helix bundles and helical hairpins. Template Assisted Synthetic Proteins (TASP).
- Beta and mixed alpha / beta constructs. Minibody and zinc finger-type.
- Design of coordination sites in de novo designed peptides. Structural sites and catalytic sites. Guided exercises in the classroom using the PyMol software.
- Towards Synthetic Biology: Artificial metalloenzymes (ARTZYMES) through redesign and de novo design. Proteins as platforms: design of metal sites with catalytic activity on the surface of proteins. Metal-Directed Protein Self-Assembly and memory of aggregation.
Introduction to laboratory activities (4 h)
- Introduction to laboratory exercises. Experiment design strategies.
Lab Activity 1. Spectrophotometric characterization and CD of serum albumin (SA) and its adducts with Cu (II) and Cu (I) - Reduction of Cu (II) / SA adduct to Cu (I) / SA - Metallochromic indicators . (4 h)
Lab Activity 2. Determination of the distribution of metal ions in a pool of solution biogues using the HySS software. (2 h)
Lab Activity 3. Study of the binding of dioxygen and carbon monoxide to myoglobin with UV-Vis absorption spectrophotometry. (4 + 4 h)
Lab Activity 4. Determination of Michaelis and Menten parameters for the oxidation reaction of horseradish peroxidase-catalyzed phenoiamine - Cysteine as inhibitor - NMR characterization of substrate and inhibitor. (4 + 4 + 4 h)
Bibliography
For the introduction to the course and to coordination compounds:
Vito Lippolis, Nelsi Zaccheroni, Vieri Fusi, Luca Giorgi, Chimica. Principi generali con esercizi, Idelson Gnocchi, 2001 (Ask the teacher)
For the sections Coordination compounds - Biological relevance, Localization and transport of metals in biological systems, Electron transfer processes, Metalloproteins and metalloenzymes:
S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry, University Science Books, 1994
D. Rehder, Bioinorganic chemistry, Oxford University Press, 2014
Handbook of metalloproteins – A. Messerschmidt (Ed.) John Wiley & Sons, 2001-2004
For the section Metal ions in neurodegenerative disorders:
Reviews and articles proposed by the teacher.
For the section Protein Design and Metalloprotein Design
V. Köhler (editor), Protein Design - Methods and Applications, Springer, 2014. Capitoli 1, 2, 3, 12, 13, 14 (Ask the teacher)
D. Voet, J.G. Voet, Biochemistry, 4 th ed., Wiley, 2011
Reviews proposed by the teacher.
Slides of the lectures presented by the teacher.
Teaching methods
Lectures of the teacher, during which the critical discussion by the students will be encouraged. Guided exercises, or addressed case studies will be presented. (4 CFU)
Practical Laboratory activities (2 CFU)
The slides used by the teacher will be uploaded weekly on the Elly website and will be downloadable upon access with personal credentials.
The slides will be used as educational material to prepare the final exam.
Assessment methods and criteria
An oral exam that will cover:
A presentation of a review or a scientific paper, chosen by the student and showed to the teacher prior the exam, that deals with a topic included in one of the following: Copper proteins, iron-heme proteins, metal ions in neurodegenerative disorders, Protein Design and Metalloprotein Design. The presentation must use a support such as Power Point.
During the test the student must demonstrate that he understands the basics of the Chemistry of coordination, with particular reference to examples taken from biological systems.
The test will end with a presentation of the results of possible group activities performed by students during the course related to Protein and Metalloprotein design.
Other information
A small part of work is planned to be carried out on a personal computer (as homework)
