Methods in Biochemical Investigation
A.Y. 2019/2020
Learning objectives
The study of proteins through protein biochemistry encompasses many fields of biology, including structural nad molecular biology, enzymology, pharmaceutical research and development, food science, plant biology, and more. The course is aiming to provide students with a thorough overview (theoretical and practical) of the most important and recent methods applied to enzymology (considering the behaviour of the single enzyme molecule as a main point of view) and to structural biology (i.e. X-ray crystallography, single-molecule Cryo-electron microscopy), focusing in particular on the scientific objectives that these techniques can help to achieve.
Expected learning outcomes
After following this course, the students will develop a better understanding of the experimental strategies applied in biological chemistry, in particular regarding protein enzymology and structural biology. At the end of this class , the students are expected to have acquired the ability to correlate the theoretical and experimental connections among the teached disciplines and their relevance to biochemical developments.
Lesson period: Second semester
Assessment methods: Esame
Assessment result: voto verbalizzato in trentesimi
Single course
This course cannot be attended as a single course. Please check our list of single courses to find the ones available for enrolment.
Course syllabus and organization
Single session
Responsible
Lesson period
Second semester
Course syllabus
The program of the Advanced Enzymology part of the course includes the following main topics: enzyme reaction mechanisms and related rate equations under steady-state conditions; one- and two-substrate enzyme reactions; reversible enzyme reactions; techniques for the study of steady-state phase of enzyme reactions; enzyme assays; determination of enzyme kinetic parameters; enzyme inhibition; experimental design and interpretation of results in the kinetic study of enzyme reactions.
The Structural Biology program is focused on the 3D structure determination and analysis of proteins by using biocrystallographic and bioinformatic techniques. Experimental techniques for protein crystal growth will be described in detailswith application to soluble and membrane proteins. X-ray diffraction on protein crystals will be treated extensively (X-ray sources, synchrotron light, atomic scattering factors, structure factors, the "phase problem", phasing by molecular replacement and heavy atoms, crystallographic refinement). Protein model validation techniques will be analyzed, together with the main features of the Protein Data Bank. In addition, the theoretical basis and the practical applications of single-molecule cryo-EM techniques will be treated.
The Structural Biology program is focused on the 3D structure determination and analysis of proteins by using biocrystallographic and bioinformatic techniques. Experimental techniques for protein crystal growth will be described in detailswith application to soluble and membrane proteins. X-ray diffraction on protein crystals will be treated extensively (X-ray sources, synchrotron light, atomic scattering factors, structure factors, the "phase problem", phasing by molecular replacement and heavy atoms, crystallographic refinement). Protein model validation techniques will be analyzed, together with the main features of the Protein Data Bank. In addition, the theoretical basis and the practical applications of single-molecule cryo-EM techniques will be treated.
Prerequisites for admission
A revision of the topics covered by the basic biochemistry and molecular biology classes included in the bachelor curriculum before attending the course is highly recommended.
Teaching methods
Teaching Mode: Classroom lectures supported by projected material with common discussions on experimental design, data analysis, and specific case studies. Demonstration of experiments on enzyme kinetics will be provided using specific computer simulation programs.
Teaching Resources
Students can refer to their text of Biochemistry for the general aspects of the structure of enzymes and catalysis. Further useful insights can be found in:
- N. Price and L. Stevens, "Fundamentals of Enzymology - Cell and Molecular Biology of Catalytic Proteins" 3rd Ed., Oxford University Press, 1999.
- A. Fersht, "Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding", WH Freeman & Co., 1999
For the part of Structural Biology, the following textbooks are suggested:
- Ducruix and R. Giegé (Oxford University Press)
- Crystallography made crystal clear. A guide for users of macromolecular models. By Gale Rhodes (Elsevier)
Copies of the slides projected in the classroom as well as other materials will be made available through the course website on the ARIEL platform of the University of Milano (https://mnardinimib.ariel.ctu.unimi.it/v5/home/Default.aspx). By no means, this material replaces the lectures or a textbook. The material is made available only to registered students of the Degree Course in Molecular Biotechnology and Bioinformatics and should not be distributed to others.
- N. Price and L. Stevens, "Fundamentals of Enzymology - Cell and Molecular Biology of Catalytic Proteins" 3rd Ed., Oxford University Press, 1999.
- A. Fersht, "Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding", WH Freeman & Co., 1999
For the part of Structural Biology, the following textbooks are suggested:
- Ducruix and R. Giegé (Oxford University Press)
- Crystallography made crystal clear. A guide for users of macromolecular models. By Gale Rhodes (Elsevier)
Copies of the slides projected in the classroom as well as other materials will be made available through the course website on the ARIEL platform of the University of Milano (https://mnardinimib.ariel.ctu.unimi.it/v5/home/Default.aspx). By no means, this material replaces the lectures or a textbook. The material is made available only to registered students of the Degree Course in Molecular Biotechnology and Bioinformatics and should not be distributed to others.
Assessment methods and Criteria
The evaluation of the student's performance will be based on a written examination with open-answer questions and exercises spanning all topics covered in the class. Each student is allowed to give the exam on the enzymology part of the program in oral form, by informing the teacher in advance.
The final grade will result from the joint evaluation of each candidate by the two teachers.
The final grade will result from the joint evaluation of each candidate by the two teachers.
BIO/10 - BIOCHEMISTRY - University credits: 6
Lessons: 48 hours
Professors:
Aliverti Alessandro, Nardini Marco
Shifts:
Professor(s)
Reception:
Monday, 08:30-12:30 (appointment required)
Dept. Biosciences, via Celoria 26, bldg C - floor 5