Ateneo - UPO

This course deals with the application of Nuclear Magnetic Resonance (NMR) techniques to the structural elucidation of biomolecules, including methods to obtain the three-dimensional structure of peptides and proteins. The course starts in the classroom by recalling the fundamentals of NMR spectroscopy; concepts and main techniques underlying biomolecular NMR will be introduced as well. In the NMR laboratory, it will be shown how to acquire and process either mono- and two-dimensional NMR spectra. Finally, in the computer room, it will be shown how to use software tools to: 1) analyse 1D/2D NMR spectra of small biomolecules and peptides; 2) work out the structure of unknown compounds; and 3) apply the sequence-specific assignment procedure to assign unambiguously the 2D-NMR spectra of polypeptides.

The lecturer will provide all slides and exercises discussed during the course.
Suggested textbooks:
• A. Randazzo “Guida Pratica alla Interpretazione di Spettri NMR”, Loghia, 2018, ISBN-8895122429. (in Italian)
• T.D.W. Claridge “High-Resolution NMR Techniques in Organic Chemistry”, 3rd Ed, 2006, Elsevier (in English)
• Joseph. P. Hornak “The Basics of NMR” 1997-2004. http://www.cis.rit.edu/htbooks/nmr/

-To provide the students with theoretical, methodological and practical knowledge of biomolecular NMR;
-To provide the students with the tools (including software resources) to enable the acquisition, processing and analysis of NMR spectra;
-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 the ability to apply the acquired knowledge to design, run and analyse NMR experiments autonomously and to judge the experiment outcome with critical sense.

Students must be familiar with basic concepts in organic chemistry, physical chemistry (NMR spectroscopy) and biochemistry (protein structure).

-Lectures on advanced NMR spectroscopy techniques, with exercises on the interpretation of NMR data (both 1D and 2D),
-NMR laboratory practice to acquire 1D/2D NMR spectra (Bruker Avance III spectrometer, 500 MHz)
-Exercises on spectral assignment and structural characterization assisted by dedicated software (computer room).

During the exercise sessions (either in the classroom and in the computer room) the students will solve resonance assignment problems and structure identification problems under the supervision of the teacher. During the practical NMR lab session, all the students will use the spectrometer under the supervision of the teacher. This allows the teacher to assess the level of learning in real time.
Students with physical disabilities, Learning Disabilities or Special
Education Needs can request specific services and tools via the "Staff
Sviluppo e Coordinamento Carriere e Servizi alle Studentesse e agli
Studenti", consulting the University webpage: https://www.uniupo.it/en/services/servicesstudents-physical-or-learning-disabilities.
Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the
tutor in charge of the course to define the examination modalities, concerning academic aspects.

The exam consists of a 2-hours written exam that evaluates the acquired knowledge and the student's ability to apply such knowledge to solve spectral problems. The written exam consists of eight questions spanning any of the arguments treated in the course. In particular, the exam includes:
- at least two questions to verify theoretical knowledge, learning ability and acquisition of a specific technical vocabulary;
- at least three question to assess the ability to apply knowledge and understanding (typically these questions address assignment problems or
structural elucidation problems);
-at least one question to assess the ability to apply knowledge and to make critical judgments (typically, this question deals with the analysis of
a set of NMR data to obtain as much as possible structural information about unknown compounds).
At the end of the course, a model exam text will be made available.

Classroom lectures (1.5 CFU):
-Fundamentals of NMR spectroscopy (nuclear spin, Larmor precession, chemical shift, scalar coupling, nuclear relaxation, nuclearOverhausereffect);
-Interpretation of 1D-NMR spectra for the elucidation of the structure of small biomolecules;
-Two-dimensional NMR spectroscopy (homonuclear and heteronuclear), including COSY, TOCSY, NOESY, HSQC, HMQC, HMBC;
-Analysis of homonuclear 2D-NMR spectra to elucidate the structure of biomolecules;
- Sequence-specific assignment for the resonance assignment of polypeptides.

NMR lab (2 CFU):
-How an NMR spectrometer works and practical aspects about the acquisition of NMR spectra (probe tuning, lock, shim, setting of acquisition parameters);
-Sample preparation (deuterated solvents);
-Acquisition and processing of 1D-NMR spectra (1H, 13C, 31P, 19F) of biomolecules and drugs (biotin, glutathione, dexamethasone-21-phosphate, unknown compounds);
-Acquisition and processing of 2D-NMR spectra of the compounds mentioned above (including 2D-COSY, 2D-TOCSY, 2D-NOESY, 2D-HSQC, 2D-HMQC, 2D-HMBC);
-Acquisition and processing of 2D-COSY, 2D-TOCSY and 2D-NOESY NMR spectra of small peptides for sequence-specific assignment.

Computer room (2.5 CFU):
-Processing of the NMR spectra of the above mentioned molecules (FT, phase correction, calibration, peak picking, integration);
-Multinuclear NMR resonance assignment;
-Computer-aided sequence-specific assignment of the peptide(s) whose spectra were acquired in the NMR lab by means of specialized software (Computer Aided Resonance Assignment - CARA);
-Identification of unknown compounds based on the analysis of 1D/2D NMR spectra.

-Theoretical and practical understanding of biomolecular NMR;
-Application of theoretical concepts for spectral interpretation and structural elucidation of biomolecules;
-Acquisition of a language that allow presenting the results of an NMR study with appropriate technical vocabulary.
-Capacity to solve problems of structural characterization by adopting the appropriate strategy and with independent thinking.
-Familiarity with bibliographic resources and software tools for further, autonomous deeper learning of specific aspects in biomolecular NMR.