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Biochemistry

Instructor: Leonid Sazanov, Martin Loose

Teaching Assistant: Catarina Alcarva, James Letts, Javier Guttierez-Fernandez

Description

 

This course provides an overview of the main aspects of biochemistry by relating molecular interactions to their effects on the organism as a whole. The organization and function of macromolecules is addressed through a discussion of their structure, and a study of their assembly into complexes responsible for specific biological processes. Topics include structure of proteins and protein complexes, enzyme kinetics and interconnection of individual proteins into tightly regulated networks.

The course will be held in three different parts: During the first four weeks, we will cover the main foundations of biochemical research (at the advanced level), in the second four weeks, students will study recent scientific papers and critically present them in front of the class, and finally we will provide hands-on experience on protein purification, biochemical assays and protein crystallization and/or structure solution. The experimental part can be held on two days with five hours lab work each.

 

Background Reading 

Non-biologists should read basic chapters from textbooks below. Biologists should update themselves on the following chapters (books available in IST library): 

Biochemistry (Voet, 4th ed.):

    Ch. 8: Three-dimensional structure of proteins
    Ch. 9: Protein folding, dynamics and structure evolution.

Molecular biology of the cell (Alberts, 5th ed.):

    Ch. 3: Proteins
    Ch. 8: Manipulating proteins, DNA and RNA, Section: Analyzing proteins (general basic info about some of used techniques).

Molecular cell biology (Lodish, 7th ed.):

    Ch. 3: Protein structure and function
    Ch. 10: Biomembrane structure, Section 10.2: Membrane proteins. Structure and basic function.
    (there is a similar chapter in Alberts).

 

Structural biology and X-ray crystallography basics online:

Crystallography 101 
www.ruppweb.org/Xray/101index.html

The Fourier Picture book and The Interactive Structure Factor Tutorial
www.ysbl.york.ac.uk/~cowtan/applets/applets.html

History:
http://onlinelibrary.wiley.com/doi/10.1111/febs.12796/full

 

Paper presentation: 

Each paper will be prepared by two students, who both present the paper. Presentations will be interactive:  presenters can ask questions to the audience; anybody can interrupt to ask something at anytime. 

Explain

Background perspective of the field that led to the question being addressed 


Main question that is addressed 


Approach used, in particular the experimental techniques 


Message of each figure: what does each piece of data show? 


In how far are the conclusions of the paper supported? 


Group discussion: starting from this paper, what is the next thing to do? 
Ask us before your presentation if anything is unclear! 


Send us an e-mail to arrange a meeting anytime 


Or drop by in the walk in hour: Fri 11-12 am either LS or ML will be available.

 

Recitations (Tuesdays, with TAs): 


Questions about previous lectures from that week 


Discussion of background for next week, questions asked of students about 
background for next week 


Students must have already read the indicated recitation reading before coming into 
the recitation 


 


Requirements/Exams

Select a paper, present, defend it, tell us why you like it, discuss strengths/potential weaknesses, tell us what you would do next; done in a team of two students; each group has 20 min to present the paper followed by 10 min for questions and discussion; no powerpoint.

 


Credits

Academic Credits: 
6 ECTS



Final Grade

25% final presentation


30% presentation in class


25% participation in class


20% participation in recitations 


 

 

Schedule (subject to change)

Lecture Date Topic Location
       
1 March 1st Welcome: Introduction of instructors, students and topics. Mondi 1
2 March 3rd The importance of in vitro reconstitution of cellular functions (Martin) Mondi 1
3 March 8th Basic of protein structure (Leonid) Mondi 1
4 March 10th ''Do not waste clean thoughts on dirty enzymes'' (Martin) Mondi 1
5 March 15th Protein complexes of the respiratory chain (Part 1) (Leonid) Mondi 1
6 March 17th Fluorescence microscopy with proteins (Martin) Mondi 1
  March 21st-April 1st 
Easter break  
7 April 5th Protein complexes of the respiratory chain (Part 2) (Leonid) Mondi 1
8 April 7th How to measure forces generated by proteins (Martin) Mondi 1
9 April 12th Hana and Mina: A6 Methods "Landau and Rosenbusch. Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14532–14535, (1996)" Mondi 1
10 April 14th Feyza and Olena: B1 Kinesin: from its discovery to measuring its step size Mondi 1
11 April 19th Huibin and Maria: A2. XFEL (first time resolved studies at high resolution) Mondi 1
12 April 21th Hana and Mina: B6. Vesicle trafficking Mondi 1
13 April 26th Julia and Kathrin: A3 Protein engineering for structure solution (Nobel prize) Mondi 1
14 April 28th Urban: B5. In vitro reconstitution of RNA polymerase transcription Mondi 1
15 May 3rd Paulo and Urban: A4. Unique mechanism (Nobel prize) Mondi 1
  May 5th No lecture - holiday Mondi 1
16 May 10th Gregory: A5. Methods "Ayyer K, et al. Macromolecular diffractive imaging using imperfect crystals. Nature. 2016 Feb 11;530(7589):202-6. doi: 10.1038/nature16949." Mondi 1
17 May 12th Julia and Kathrin: B4. Reconstitution of actin-based motility Mondi 1
18 May 17th Feyza and Olena: A1 Single Molecule Cryo-EM "Bai, X.-C., Fernandez, I. S., McMullan, G., & Scheres, S. H. W. (2013). Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife, 2, e00461. doi.org/10.7554/eLife.00461" Mondi 1
19 May 19th Paulo and Maria: B3. Spatial regulation of bacterial cell division Mondi 1
20 May 24th Gregory and Huibin: B2. Studying the mitotic spindle in vitro Mondi 1
  May 31st-June 14th 
Practicals  

Lecture content:


March 3rd: The importance of in vitro reconstitution for biological research

In the first lecture, we will discuss the following questions:

 
- Why would one want to reconstitute cellular behavior?
- Why is it possible to reconstitute cellular functions?
- What needs to be considered for in vitro reconstitution experiments? 
 

These papers will be useful for the discussion:

Liu, A. P., & Fletcher, D. A. (2009). Biology under construction: in vitro reconstitution of cellular function. Nature Reviews Molecular Cell Biology, 10(9), 644–650. doi.org/10.1038/nrm2746

Hartwell, L. H., Hopfield, J. J., Leibler, S., & Murray, A. W. (1999). From molecular to modular cell biology. Nature, 402(6761 Suppl), C47–C52. doi.org/10.1038/35011540

Vahey, M. D., & Fletcher, D. A. (2014). The biology of boundary conditions: cellular reconstitution in one, two, and three dimensions. Current Opinion in Cell Biology, 26, 60–68. doi.org/10.1016/j.ceb.2013.10.001

 


March 8th: 
Basics of protein structure 

Topics from primary to quaternary structure of proteins will be covered, along with two methods of structure determination: X-ray crystallography and cryo-EM.

Reading: background reading from textbooks listed in the introduction.

 


March 10th: 
Protein purification

Papers discussed in lecture:

Petek, N. A., & Mullins, R. D. (2014). Bacterial actin-like proteins: purification and characterization of self-assembly properties. Methods in Enzymology, 540, 19–34. doi.org/10.1016/B978-0-12-397924-7.00002-9

Kornberg, A. (2003). Ten commandments of enzymology, amended. Trends Biochem Sci, 28(10), 515–517. doi.org/10.1016/j.tibs.2003.08.007

Castoldi, M., & Popov, A. V. (2003). Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expression and Purification, 32(1), 83–88. doi.org/10.1016/S1046-5928(03)00218-3

Gräslund, S., Nordlund, P., Weigelt, J., Bray, J., Gileadi, O., Knapp, S., et al. (2008). Protein production and purification. Nature Methods, 5(2), 135–146. doi.org/10.1038/nmeth.f.202


March 15th: Protein complexes of the respiratory chain.

Structure and function of respiratory complexes I and III will be covered.

Reading:

Reviews:

[1] C.C. Moser, T.A. Farid, S.E. Chobot, P.L. Dutton, Electron tunneling chains of mitochondria, Biochim. Biophys. Acta, 1757 (2006) 1096-1109.

[2] L.A. Sazanov, A giant molecular proton pump: structure and mechanism of respiratory complex I, Nature reviews. Molecular cell biology, 16 (2015) 375-388.

[3] U. Brandt, Energy converting NADH:quinone oxidoreductase (complex I), Annu. Rev. Biochem., 75 (2006) 69-92.

[4] M. Wikstrom, V. Sharma, V.R. Kaila, J.P. Hosler, G. Hummer, New perspectives on proton pumping in cellular respiration, Chem Rev, 115 (2015) 2196-2221.

[5] D. Xia, L. Esser, W.K. Tang, F. Zhou, Y. Zhou, L. Yu, C.A. Yu, Structural analysis of cytochrome bc1 complexes: implications to the mechanism of function, Biochim. Biophys. Acta, 1827 (2013) 1278-1294.

 

Original articles:

  [6] R. Baradaran, J.M. Berrisford, G.S. Minhas, L.A. Sazanov, Crystal structure of the entire respiratory complex I, Nature, 494 (2013) 443-448.

 [7] V. Zickermann, C. Wirth, H. Nasiri, K. Siegmund, H. Schwalbe, C. Hunte, U. Brandt, Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I, Science, 347 (2015) 44-49.

[8] K.R. Vinothkumar, J. Zhu, J. Hirst, Architecture of mammalian respiratory complex I, Nature, (2014).

 [9] S. Iwata, J.W. Lee, K. Okada, J.K. Lee, M. Iwata, B. Rasmussen, T.A. Link, S. Ramaswamy, B.K. Jap, Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex, Science, 281 (1998) 64-71.

[10] Z.L. Zhang, L.S. Huang, V.M. Shulmeister, Y.I. Chi, K.K. Kim, L.W. Hung, A.R. Crofts, E.A. Berry, S.H. Kim, Electron transfer by domain movement in cytochrome bc1, Nature, 392 (1998) 677-684.

 


March 17th: 
Fluorescence microscopy with proteins

Some useful information about using fluorescence to study protein properties:

The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling 

 


April 5th: Protein complexes of the respiratory chain (Part 2).

Structure and function of respiratory complexes IV and V will be covered.

Reading:

Reviews:

 [1] M. Wikstrom, M.I. Verkhovsky, Towards the mechanism of proton pumping by the haem-copper oxidases, Biochim. Biophys. Acta, 1757 (2006) 1047-1051.

[2] P. Brzezinski, P. Adelroth, Design principles of proton-pumping haem-copper oxidases, Curr. Opin. Struct. Biol., 16 (2006) 465-472.

[3] J.E. Walker, The ATP synthase: the understood, the uncertain and the unknown, Biochem Soc Trans, 41 (2013) 1-16.

 

Original articles:

 [4] T. Tsukihara, H. Aoyama, E. Yamashita, T. Tomizaki, H. Yamaguchi, K. Shinzawa-Itoh, R. Nakashima, R. Yaono, S. Yoshikawa, The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A, Science, 272 (1996) 1136-1144.

[5] S. Iwata, C. Ostermeier, B. Ludwig, H. Michel, Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans, Nature, 376 (1995) 660-669.

[6] I.N. Watt, M.G. Montgomery, M.J. Runswick, A.G. Leslie, J.E. Walker, Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria, Proc. Natl. Acad. Sci. U S A, 107 (2010) 16823-16827.

[7] M. Allegretti, N. Klusch, D.J. Mills, J. Vonck, W. Kuhlbrandt, K.M. Davies, Horizontal membrane-intrinsic alpha-helices in the stator a-subunit of an F-type ATP synthase, Nature, 521 (2015) 237-240.

 


April 7th: 
How to measure forces generated by proteins

Neuman, K. C., & Nagy, A. (2008). Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods, 5(6), 491–505. doi.org/10.1038/nmeth.1218

 

 


Starting from April 12th: 
Paper discussions

A. Tuesdays: Structural Biology

April 12th: Hana and Mina: A6. Methods

Landau and Rosenbusch. Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14532–14535, (1996)

April 19th: Huibin and Maria: A2. XFEL (first time resolved studies at high resolution)

Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein.  Science. 2014 Dec 5;346(6214):1242-6. doi: 10.1126/science.1259357. 

April 26th: Julia and Kathrin: A3. Protein engineering for structure solution (Nobel prize)

Kobilka, B. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex.

Nature. 2011 Jul 19;477(7366):549-55. doi: 10.1038/nature10361.

May 3rd: Paulo and Urban: A4. Unique mechanism (Nobel prize)

Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.

Abrahams JP, Leslie AG, Lutter R, Walker JE. Nature. 1994 Aug 25;370(6491):621-8.

May 10th: Gregory: A5. Methods

Ayyer K, et al. Macromolecular diffractive imaging using imperfect crystals. Nature. 2016 Feb 11;530(7589):202-6. doi: 10.1038/nature16949.

May 17th: Feyza and Olena: A1. Single Molecule Cryo-EM

(first demonstration of the power of Direct Electron Detectors and statistical processing)

Bai, X.-C., Fernandez, I. S., McMullan, G., & Scheres, S. H. W. (2013). Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife, 2, e00461. doi.org/10.7554/eLife.00461

 

B. Thursdays: In vitro reconstitution

For each topic, I picked a couple of papers to illustrate how scientific research progresses. Papers in italics represent additional information, that should be mentioned in the presentation. Non-italic papers are the actual paper to be presented. 

April 14th: Feyza and Olena: B1. Kinesin: from its discovery to measuring its step size

Vale, R. D., Reese, T. S., & Sheetz, M. P. (1985). Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell, 42(1), 39–50.

Svoboda, K., Schmidt, C. F., Schnapp, B. J., & Block, S. M. (1993). Direct observation of kinesin stepping by optical trapping interferometry. Nature, 365(6448), 721–727. doi.org/10.1038/365721a0

Yildiz, A., Tomishige, M., Vale, R. D., & Selvin, P. R. (2004). Kinesin walks hand-over-hand. Science, 303(5658), 676–678. doi.org/10.1126/science.1093753

April 21st: Hana and Mina: B6. Vesicle trafficking

Orci, L., Glick, B. S., & Rothman, J. E. (1986). A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell, 46(2), 171–184.

Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S. Y., Hamamoto, S., Schekman, R., & Yeung, T. (1998). COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell, 93(2), 263–275.

Zanetti, G., Prinz, S., Daum, S., Meister, A., Schekman, R., Bacia, K., & Briggs, J. A. G. (2013). The structure of the COPII transport-vesicle coat assembled on membranes. eLife, 2, e00951. doi.org/10.7554/eLife.00951

April 28th: Urban: B5. In vitro reconstitution of RNA polymerase transcription

Sayre, M. H., Tschochner, H., & Kornberg, R. D. (1992). Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae. J Biol Chem, 267(32), 23376–23382.

Wang, M. D., Schnitzer, M. J., Yin, H., Landick, R., Gelles, J., & Block, S. M. (1998). Force and velocity measured for single molecules of RNA polymerase. Science, 282(5390), 902–907.

May 12th: Julian and Kathrin: B4. Reconstitution of actin-based motility

Theriot, J. A., Rosenblatt, J., Portnoy, D. A., Goldschmidt-Clermont, P. J., & Mitchison, T. J. (1994). Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts. Cell, 76(3), 505–517.

Loisel, T. P., Boujemaa, R., Pantaloni, D., & Carlier, M. F. (1999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature, 401(6753), 613–616. doi.org/10.1038/44183

May 19th: Paulo and Maria: B3. Spatial regulation of bacterial cell division

Loose, M., Fischer-Friedrich, E., Ries, J., Kruse, K., & Schwille, P. (2008). Spatial regulators for bacterial cell division self-organize into surface waves in vitro. Science, 320(5877), 789–792. doi.org/10.1126/science.1154413

Zieske, K., & Schwille, P. (2014). Reconstitution of self-organizing protein gradients as spatial cues in cell-free systems. eLife, 3. doi.org/10.7554/eLife.03949

May 24th: Gregory and Huibin: B2. Studying the mitotic spindle in vitro

Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A., & Karsenti, E. (1996). Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature, 382(6590), 420–425. doi.org/10.1038/382420a0

Brugués, J., Nuzzo, V., Mazur, E., & Needleman, D. J. (2012). Nucleation and transport organize microtubules in metaphase spindles. Cell, 149(3), 554–564. doi.org/10.1016/j.cell.2012.03.027

Z

 

 

Recitations

March 15th - The folding problem in proteins

Assessing the quality of 3D structures

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