“Advanced Methods in Biochemistry” is a fundamentally practical subject designed to consolidate the theoretical foundations acquired in the previous courses of the degree. It will allow you to learn how to use spectroscopic techniques in the study of biomolecule structures and intermolecular interactions. In addition, you will deepen the knowledge of protein-cell membrane interactions, as the spectroscopic studies will be complemented with immunochemical techniques and assays in eukaryotic cells. The theoretical contents of the course focus on X-ray crystallography, and on the physical and mathematical foundations behind it. This high-resolution technique allows determining the structures of biomolecules, mainly proteins, at atomic level.



“Advanced Methods in Biochemistry” is directly linked to “Spectroscopy of Biomolecules” (3rd year), since it involves the practical development of the theoretical foundations previously learned in it. It is also connected to “Structural Biology: Biomedical applications” (4th year), as it allows to deepen the knowledge of protein structure. Finally, the practical sessions are oriented to understand the signaling processes originated after the protein-cell membrane interaction, which strongly complements “Cellular Signaling” (2nd year).



In this subject, you will study and experience the principles, instrumentation and applications of the spectroscopic techniques commonly used in research and diagnostic laboratories. This will allow you to interpret the processes that take place in biological systems and will train you for your future professional practice.

- Know the principles, the instrumentation and the applications of the main techniques used in Biochemistry and Molecular Biology

- Gain ability to separate isolated substances from living cells, and determine their structures and chemical and functional properties

- Interpret the results obtained by spectroscopic techniques in terms of dynamic conformation of biomolecules

- Protein conformational stability: Denaturation of a protein with different agents. Variation of Gibbs free energy of the process. Cm comparison

- FT-IR spectrum analysis: Secondary structure of proteins

- Analysis of circular dichroism spectra of macromolecules: secondary structure of proteins and nucleic acids

- Effect of the environmental polarity on the fluorescence spectrum: Emission properties of free probes and probes localized/bound to biomolecules

- Protein-ligand interaction: Kd of the binding by fluorescent parameters. Anisotropy, extinction, life times. Characterization of hormonal receptors

- Detection of Ca2+ by fluorescence: Kd and kinetics

- FRET: Calculation of intermolecular distances

- Protein detection by immunofluorescence.

- Cell viability analysis.

- Applications of fluorescence polarization: experiments related to the technique

- Analysis of Nuclear Magnetic Resonance Spectra: 3D resolution of protein structure

-Introduction to X-ray diffraction 3D resolution of protein structure

The master classes (M) we will be used to explain the information that was not included in the course "Spectroscopy of Biomolecules" as well as to explain the practice protocols.



The contents of the subject are also worked throughout laboratory practices (PL) and computer practices (PO), where you will process your own data obtained in the laboratory or real experimental data we will provide you. In another set of PO you will perform in silico practices in order to become familiar with instrumentation not accessible to the laboratory.



Finally, you will be asked to prepare and present seminars where you will explain more complex or recent techniques of your choice to other students in the class.

• Final Assessment System
• Tools and qualification percentages:
  • Team projects (problem solving, project design)) (%): 100

According to current regulations, students have the right to renounce the continuous evaluation system and to be assessed by a final exam. To do so, they should communicate it in writing to the faculty responsible for the subject, in a period of time that will never be longer than 9 weeks from the beginning of the semester.



If a student wants to renounce the ordinary call it will be enough not to take the final exam on the official date, so that she/he will have the final mark “No Presentado” (“No show”).



The evaluation system will be as follows:

- Practical work (PL + PO) and final report of the laboratory and computer practices: 75%

- Individual work and seminars on a current topic related to the contents of the subject: 10%

- Crystallography (theoretical exam + computer practices): 15%



These sections will be evaluated according to the following criteria:

- Adequate implementation of the practice protocol; correct analysis, interpretation and presentation of the results obtained

- Correct approach and development of the exercises; elaboration and presentation of the entrusted tasks

- Attendance to laboratory and computer practices is mandatory and will be subject to control



The final mark will be obtained by adding the partial marks obtained in each section. In order to be able to pass the subject, it is necessary to obtain 40% of the maximum mark in each of the aforementioned sections. The subject will be considered as passed when the sum of all the partial marks reaches a final mark of 5 out of 10.



If the student doesn´t pass the subject in the ordinary call, she/he will NOT have to repeat those sections with marks above the threshold of 40% in the extraordinary call of the current year (July).

Students will have the right to be evaluated through the final evaluation system, regardless of whether or not they have participated in the continuous evaluation system. To do this, students must submit in writing to the teaching staff responsible for the subject the waiver of continuous assessment, for which they will have a period of 9 weeks from the beginning of the semester



If a student wants to renounce the extraordinary call it will be enough not to take the final exam on the official date, so that she/he will have the final mark “No Presentado” (“No show”).

Moodle page of the course: http://moodle3.ehu.es/course/view.php?id=2652
In that link, you will find the practice protocols posted. You must carefully read these protocols before you go to the practical session, and bring them with you to the lab.
The moodle plataform will also be used for the delivery, correction and evaluation of the reports and work that you will be asked to present during the course.

Basic bibliography

- Estructura de proteínas. Gómez-Moreno C, Sancho J (2003) Ed. Ariel Ciencia

- Biological Spectroscopy. Campbell ID, Dwek RA (1984) Benjamin Cummings

- Methods in Molecular Biophysics. Structure, dynamics, function. Serdyuk IN, Zaccai NR, Zaccai J (2007) Cambridge

In-depth bibliography

- Principles of Physical Biochemistry. van Holde KE, Johson WC, Shing PH (2006) 2nd ed. Prentice Hall
- Physical Chemistry. Principles and Applications in Biological Sciences. Tinoco I, Sauer K, Wang JC (2001) 4th ed. Prentice Hall
- Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology. Rupp B (2010) Garland Sciences
- Spectroscopy for the Biological Sciences. Hammes GG (2005) Wiley Interscience
- Principles of Fluorescence Spectroscopy. Lakowicz JR (2006) 3rd ed. Springer
- Molecular Spectroscopy. Brown JM (1998) Oxford University Press
- Introduction to Biophysical Methods for Protein and Nucleic Acid Research. Glasel JA,
Deutscher MP (1995) Academic Press
- Biophysical Chemistry. Part II: Techniques for the study of biological structure and function. Cantor CR, Schimmel PR (1980) W. H. Freeman and Company.

Journals

- Nature
- Nature Methods
- Annual Review o0f Biophysics
- Biophysical Journal
- Biochemistry

Web addresses

X-ray crystallography
http://www.ruppweb.org/Xray/101index.html
http://www.mpibpc.mpg.de/groups/de_groot/compbio/p3/index.html#x-ray
http://www.biop.ox.ac.uk/coot/
http://www.ysbl.york.ac.uk/%7Ecowtan/sfapplet/sfintro.html

NMR:
http://nmrwiki.org/wiki/index.php?title=Materials_for_teaching_NMR

Circular dichroism:
http://pcddb.cryst.bbk.ac.uk/home.php
http://biomodel.uah.es/lab/dc/inicio.htm