Ateneo - UPO

This course is about the application of Nuclear Magnetic Resonance (NMR) techniques in structural biology, including the study of protein/ligand interactions. The course starts by introducing the fundamental concepts of 1D/2D NMR spectroscopy and their application to the analysis of the NMR spectra of small biomolecules/drugs. Then, the basic NMR techniques used to study peptides and proteins will be introduced, namely: i) the sequence-specific method for the assignment of the protein backbone; ii) methods to determine the three-dimensional structure of peptides and proteins from NMR-derived data; and iii) methods to study protein/ligand interactions.

The lecturer will provide all slides and exercises discussed during the course. Suggested textbooks:
• T.D.W. Claridge “High-Resolution NMR Techniques in Organic Chemistry”, 3rd Edition, Elsevier Science, 2016.
• H. Friebolin “Basic one- and two-dimensional NMR spectroscopy”, VCH, 1993.
• Joseph. P. Hornak “The Basics of NMR” 1997-2004. http://www.cis.rit.edu/htbooks/nmr/ (online book)

- To provide the students with basic concepts of biomolecular NMR;
- To provide the students with conceptual tools enabling the interpretation of NMR spectra and of protein/ligand interaction data;
- To create awareness about the potential (and limitations) of NMR spectroscopy in regards to the resolution of the three-dimensional structure of biomolecules;
- To provide the students with a suitable technical vocabulary in relation to the topics described in the course;
- To foster the capacity of autonomous further learning.

Students have to be familiar with basic concepts in Organic Chemistry, Physical Chemistry (spectroscopy) and Biochemistry (protein structure).

Classroom lectures integrated with exercises about spectral interpretation, with critical discussion of the results.

The main topics of the course will be discussed collectively in the classroom and the knowledge will be applied to work out spectral interpretation problems, to assess the level of understanding in real time.

See the description of the general course.

NMR methods to assess the three dimensional structure of proteins and to study protein/ligand interactions.
- Basic concepts of NMR: nuclear spins; nuclei in a magnetic field; nuclear spin energy levels and populations; Larmor frequency; macroscopic magnetization; the resonance condition; multinuclear NMR.
- Pulse (FT) NMR: the vector model of NMR; rotating frame; radiofrequency pulses; flip angle; Free Induction Decay (FID); time and frequency domains; Fourier transform (FT); pulse sequences.
- The NMR spectrum: chemical shift; scalar coupling; signal area; 1H and 13C-NMR spectroscopy; problems and exercises about the interpretation of 1D NMR spectra.
- Nuclear relaxation: longitudinal relaxation (T1); transverse relaxation (T2); dipolar coupling; molecular motions and their effect on nuclear relaxation; the nuclear Overhauser effect (NOE) and internuclear distances.
- Chemical exchange: exchange regimes (slow, intermediate, fast); effects of chemical exchange on the NMR spectrum and on nuclear relaxation; the protein/ligand association equilibrium as a chemical exchange process; methods to study protein/ligands interactione based on the observation of the ligand; Saturation Transfer Difference (STD) NMR; Group Epitope Mapping (GEM); water-LOGSY; measurement of dissociation constants; NMR screening in drug discovery.
- 2D-NMR spectroscopy: 2D-COSY, 2D-TOCSY, 2D-NOESY and 2D-HSQC spectra; examples and exercises about the interpretation of 2D NMR spectra.
- Multidimensional NMR techniques to assess the three-dimensional structure of peptides and proteins: the sequence-specific assignment method; NOE-based methods to measure internuclear distances; elements of molecular dynamics calculations.
- Methods to study protein/ligand interactions based on the observation of the protein: 2D 1H,15N-HSQC spectra; Chemical Shift Perturbation (CSP) mapping; SAR by NMR.

-Basic knowledge and understanding of: i) the theoretical principles of NMR spectroscopy; ii) the NMR techniques for the determination of protein structure; and iii) the NMR techniques to study protein/ligand interactions.
-Ability to apply knowledge and understanding to: i) the interpretation of NMR spectra (spectral assignment and/or prediction of simple molecules spectra); and ii) the use of NMR data to study protein/ligand interactions.
-Autonomy of judgment: ability to evaluate critically the possibilities and the limitations of NMR techniques to assess the structure and dynamics of biomolecules and protein/ligand complexes.
- Communication skills: acquisition of technical language and vocabolary enabling to present an NMR study in a clear, rigorous, and concise manner.
- Learning skills: ability to use educational resources and literature references, in an autonomous manner, to gain further insights into specific NMR techniques for the assessment of protein structure and interaction with ligands.