Chemistry

Structures and polymorphism of the most important biological macromolecules: proteins, nucleic acids and association complexes proteins-nucleic acids, proteins/oligosaccharides, proteins/lipids (summary). (2h) Principles of spectroscopy: interactions between radiation and matter (summary). (2 h) Electronic spectroscopy in absorption – electronic bands, Hamiltonians separable in σ and π bonds. Exciton splitting in biopolymers. Electronic spectra in proteins and nucleic acids. Hypochromism in DNA: evaluation of melting temperature, ∆H and ∆S of DNA in various experimental conditions. (3 h) Vibrational spectroscopy – Normal modes of vibration, functional groups. Vibrational spectra of nucleobases of nucleic acids, hydrogen bond effect and determination of Watson and Crick association among nucleobases. Investigation of secondary structure in proteins and peptides: amide-I and amide-II vibrational bands. Analysis of vibrational spectra of proteins to estimate the secondary structures in proteins. (3 h) Circular dichroism in polypeptides, proteins with linear structures and double stranded nucleic acids. Circular dichroism spectroscopy - Optical activity. Physics of circular dichroism. Circularly polarized electromagnetic radiation. Ellipticity and optical rotatory dispersion. Rotatory strength. Exciton splitting. Cotton effect. Conservative CD bands. Circular dichroism in proteins and nucleic acids. Analysis (single value decomposition) of protein spectra to evaluate the fraction of secondary structures in proteins. Circular dichroism of quadruplex structures of nucleic acids. Circular dichroism spectroscopy to investigate the interactions of minor goove ligands and intercalants and DNA. (12 h) Fluorescence spectroscopy – Ground state, singlet and triplet state. Fluorescence and phosphorescence emission. Emission lifetime. Vibronic bands for absorption and fluorescence. Fluorescence instrumentation. Fluorophores in proteins and nucleic acids. Fluorescent probes. Solvent effects. Fluorescence decay and quantum yield. Fluorescence resonance energy transfer. Fluorescence linear polarization and anisotropy. Fluorescence of proteins and globular structure. Fluorescence spectroscopy to investigate protein assembling. Determination of β secondary structures (fibril aggregation) in amyloids by thioflavin T assay. Interactions of double stranded DNA with minor groove ligands and intercalants by fluorescence measurements. (12 h) Nuclear Magnetic Resonance Spectroscopy - Definition and properties of linear momentum and angular momentum in classical and quantum-mechanical approach. Electron spin, proton spin and neutron spin. Definition and properties of the magnetic moment in classical and quantum-mechanical approach. Comparison between angular momentum and magnetic moment in classical and quantum-mechanical approach. Gyromagnetic ratio. Behavior of a nucleus in a magnetic field: classical approach. Larmor's precession. Larmor's equation. Resonance phenomenon. Resonance conditions. Properties of nuclei systems. Macrosopic magnetization. Phenomenological equations of Bloch. Saturation factor. The nuclei system and the surrounding interaction. Rotating frame. Magnetic susceptibility. Shielding factor. Lamb equation. Ramsey equation. Diamagnetic and paramagnetic shielding. NMR instrumentation: definition of resolution, continuous wave method, pulse method. Characteristics of an optimal pulse. Signal processing: Fourier transform method (FT). Shape of the bands in liquids and solids. Definition of signal-to-noise ratio. Theorems and properties of the FT. Increase of the signal / noise ratio and resolution. NMR parameters. Chemical shift: fields generated by electrons around the nuclei. Shielding factor. Shielding effects and deshielding. Definition of parts per million (ppm). Chemical shift reference substances in water and in organic solvents. Dependence of the chemical shift from the nature of the substituents. Anisotropy effect of the chemical shift. Chemical shift and hydrogen bonding. Scale of chemical shift. Dependence of chemical shift from solvent, concentration, pH, ionic strength. Coupling constant: origins of the spin-spin coupling, methods of coupling transmission, types of homo- and heteronuclear coupling. Dependence on structural parameters. Karplus equation. Application to different classes of compounds. Spin systems of the first order and higher order. Chemical equivalence. Magnetic equivalence. Spin-lattice and spin-spin relaxation times. Sequences for measuring relaxation times. Relation of relaxation times with the molecular structure. Homo- and heteronuclear decoupling techniques. Homonuclear and heteronuclear Nuclear Overhauser Effect. NMR spectroscopy of C-13. Chemical shift, coupling C-13 - H-1. Decoupling techniques. Polarization transfer. SPT (selective population transfer) sequences: saturation and inversion. Two-dimensional NMR spectroscopy. Structure of a two-dimensional pulse sequence. Two-dimensional homo- and heteronuclear spectroscopy. Two-dimensional spectroscopy of homonuclear correlation COSY, DQF -COSY, TOCSY. 2D heteronuclear spectroscopy. Correlations through space: 2D spectroscopy NOESY and ROESY: theory and applications. Applications of NMR spectroscopy to biological systems: investigation of base stacking in DNA and determination of structures of proteins, peptides and nucleic acids. (32 h)

Knowledge of basic concept of magnetism, quantum mechanics and principles of spectroscopy (interaction of electromagnetic radiation with matter: time-dependent perturbations theory; emission and absorption theory, Einstein coefficients). Furthermore, composition and structure of biological macromolecules (mainly, peptides, proteins and nucleic acids) should be known.

1. Principles of Physical Biochemistry – Kensal E. van Holde, W. Curtis Johnson, P. Shing Ho, Second Edition, Prentice Hall (available at the library G. Illuminati of Chemistry Department) 2. H. Friebolin, Basic one- and two-dimensional NMR spectroscopy, Fifth Edition, Wiley VCH 3. J. R. Lakowicz, Principles of Fluorescence Spectroscopy 3rd, 2006, Springer 4.. Slides shown during the lessons and notes

The course will be held in classroom. It is composed of seventy-two hours devoted to the description of the most important spectroscopies (principles and applications). A significant number of applications to investigate proteins, nucleic acids, peptides and association complexes DNA-proteins and biological macromolecules-ligands will be presented.

Lesson attendance is not mandatory but strongly recommended

The student will be evaluated by an oral exam in which the physico-chemical principles of the most important spectroscopic techniques and applications methodologies to biological systems will be discussed. The capability of analysis, making judgment and communication will be also evaluated. Simple but exemplary systems will be proposeded to evaluate the student skills to frame the chemical problem in the correct context and choose the most suitable methodologies of investigation.

To a deeper knowledge of some issues: Biophysical Chemistry, second volume: Techniques for the Study of Biological Structure and Function; H. Friebolin: Basic one- and two-dimensional NMR spectroscopy, V Edition, Wiley VCH K. Wüthich: NMR of proteins and nucleic acids, John Wiley and Sons, Inc. Some articles published in international journals that will be discussed during lessons

The course will be held in classroom. It is composed of seventy-two hours devoted to the description of the most important spectroscopies (principles and applications). A significant number of applications to investigate proteins, nucleic acids, peptides and association complexes DNA-proteins and biological macromolecules-ligands will be presented.

Structures and polymorphism of the most important biological macromolecules: proteins, nucleic acids and association complexes proteins-nucleic acids, proteins/oligosaccharides, proteins/lipids (summary). Principles of spectroscopy: interactions between radiation and matter (summary). Electronic spectroscopy in absorption – electronic bands, Hamiltonians separable in σ and π bonds. Exciton splitting in biopolymers. Electronic spectra in proteins and nucleic acids. Hypochromism in DNA: evaluation of melting temperature, ∆H and ∆S of DNA in various experimental conditions. Vibrational spectroscopy – Normal modes of vibration, functional groups. Vibrational spectra of nucleobases of nucleic acids, hydrogen bond effect and determination of Watson and Crick association among nucleobases. Investigation of secondary structure in proteins and peptides: amide-I and amide-II vibrational bands. Analysis of vibrational spectra of proteins to estimate the secondary structures in proteins. Circular dichroism in polypeptides, proteins with linear structures and double stranded nucleic acids. Circular dichroism spectroscopy - Optical activity. Physics of circular dichroism. Circularly polarized electromagnetic radiation. Ellipticity and optical rotatory dispersion. Rotatory strength. Exciton splitting. Cotton effect. Conservative CD bands. Circular dichroism in proteins and nucleic acids. Analysis (single value decomposition) of protein spectra to evaluate the fraction of secondary structures in proteins. Circular dichroism of quadruplex structures of nucleic acids. Circular dichroism spectroscopy to investigate the interactions of minor goove ligands and intercalants and DNA. (12 h) Fluorescence spectroscopy – Ground state, singlet and triplet state. Fluorescence and phosphorescence emission. Emission lifetime. Vibronic bands for absorption and fluorescence. Fluorescence instrumentation. Fluorophores in proteins and nucleic acids. Fluorescent probes. Solvent effects. Fluorescence decay and quantum yield. Fluorescence resonance energy transfer. Fluorescence linear polarization and anisotropy. Fluorescence of proteins and globular structure. Fluorescence spectroscopy to investigate protein assembling. Determination of β secondary structures (fibril aggregation) in amyloids by thioflavin T assay. Interactions of double stranded DNA with minor groove ligands and intercalants by fluorescence measurements. Nuclear Magnetic Resonance Spectroscopy - Definition and properties of linear momentum and angular momentum in classical and quantum-mechanical approach. Electron spin, proton spin and neutron spin. Definition and properties of the magnetic moment in classical and quantum-mechanical approach. Comparison between angular momentum and magnetic moment in classical and quantum-mechanical approach. Gyromagnetic ratio. Behavior of a nucleus in a magnetic field: classical approach. Larmor's precession. Larmor's equation. Resonance phenomenon. Resonance conditions. Properties of nuclei systems. Macrosopic magnetization. Phenomenological equations of Bloch. Saturation factor. The nuclei system and the surrounding interaction. Rotating frame. Magnetic susceptibility. Shielding factor. Lamb equation. Ramsey equation. Diamagnetic and paramagnetic shielding. NMR instrumentation: definition of resolution, continuous wave method, pulse method. Characteristics of an optimal pulse. Signal processing: Fourier transform method (FT). Shape of the bands in liquids and solids. Definition of signal-to-noise ratio. Theorems and properties of the FT. Increase of the signal / noise ratio and resolution. NMR parameters. Chemical shift: fields generated by electrons around the nuclei. Shielding factor. Shielding effects and deshielding. Definition of parts per million (ppm). Chemical shift reference substances in water and in organic solvents. Dependence of the chemical shift from the nature of the substituents. Anisotropy effect of the chemical shift. Chemical shift and hydrogen bonding. Scale of chemical shift. Dependence of chemical shift from solvent, concentration, pH, ionic strength. Coupling constant: origins of the spin-spin coupling, methods of coupling transmission, types of homo- and heteronuclear coupling. Dependence on structural parameters. Karplus equation. Application to different classes of compounds. Spin systems of the first order and higher order. Chemical equivalence. Magnetic equivalence. Spin-lattice and spin-spin relaxation times. Sequences for measuring relaxation times. Relation of relaxation times with the molecular structure. Homo- and heteronuclear decoupling techniques. Homonuclear and heteronuclear Nuclear Overhauser Effect. NMR spectroscopy of C-13. Chemical shift, coupling C-13 - H-1. Decoupling techniques. Polarization transfer. SPT (selective population transfer) sequences: saturation and inversion. Two-dimensional NMR spectroscopy. Structure of a two-dimensional pulse sequence. Two-dimensional homo- and heteronuclear spectroscopy. Two-dimensional spectroscopy of homonuclear correlation COSY, DQF -COSY, TOCSY. 2D heteronuclear spectroscopy. Metabolomics. Correlations through space: 2D spectroscopy NOESY and ROESY: theory and applications. Applications of NMR spectroscopy to biological systems: investigation of base stacking in DNA and determination of structures of proteins, peptides and nucleic acids.

Knowledge of basic concept of magnetism, quantum mechanics and principles of spectroscopy (interaction of electromagnetic radiation with matter: time-dependent perturbations theory; emission and absorption theory, Einstein coefficients). Furthermore, composition and structure of biological macromolecules (mainly, peptides, proteins and nucleic acids) should be known.

1. Principles of Physical Biochemistry – Kensal E. van Holde, W. Curtis Johnson, P. Shing Ho, Second Edition, Prentice Hall (available at the library G. Illuminati of Chemistry Department) 2. H. Friebolin, Basic one- and two-dimensional NMR spectroscopy, Fifth Edition, Wiley VCH 3. J. R. Lakowicz, Principles of Fluorescence Spectroscopy 3rd, 2006, Springer 4.. Slides shown during the lessons and notes

Attendance is not mandatory but strongly recommended.

The student will be evaluated by an oral exam in which the physico-chemical principles of the most important spectroscopic techniques and applications methodologies to biological systems will be discussed. The capability of analysis, making judgment and communication will be also evaluated. Simple but exemplary systems will be proposeded to evaluate the student skills to frame the chemical problem in the correct context and choose the most suitable methodologies of investigation.

To a deeper knowledge of some issues: Biophysical Chemistry, second volume: Techniques for the Study of Biological Structure and Function; H. Friebolin: Basic one- and two-dimensional NMR spectroscopy, V Edition, Wiley VCH K. Wüthich: NMR of proteins and nucleic acids, John Wiley and Sons, Inc. Some articles published in international journals that will be discussed during lessons

The course will be held in classroom. It is composed of seventy-two hours devoted to the description of the most important spectroscopies (principles and applications). A significant number of applications to investigate proteins, nucleic acids, peptides and association complexes DNA-proteins and biological macromolecules-ligands will be presented.