2. Denaturácia a renaturácia RNázy A Nobelova cena z chémie v roku 1972 za práce o zvinovaní proteínov

4. Anfinsen Experiment Denaturation of ribonuclease A (4 disulfide bonds), with 8 M Urea containing  - mercaptoethanol, leads to random coil and no activity

5. Anfinsen Experiment After renaturation, the refolded protein has native activity, despite 105 ways to renature the protein. Conclusion: All the information necessary for folding into its native structure is contained in the amino acid sequence of the protein.

6. Anfinsen Experiment Remove  -mercaptoethanol only, oxidation of the sulfhydryl group, then remove urea → scrambled protein, no activity Further addition of trace amounts of  -mercaptoethanol converts the scrambled form into native form. Conclusion: The native form of a protein has the thermodynamic- ally most stable structure.

7. Models of Protein Folding

8. Framework model of protein folding Supported by experimental observation of rapid formation of secondary structure during protein folding process N C

9. Framework model of protein folding N C Formation of individual secondary structure elements

10. Framework model of protein folding N C Coalescence and rearrangement of individual secondary structure elements

11. Nuclear condensation model N C Supported by protein engineering studies and various theoretical calculations

12. Nuclear condensation model N C Formation of a nucleus of hydrophobic residues

13. Nuclear condensation model N C Expansion of nucleus

14. Steps of Folding Unfolded  bury core  2 o  Molten globule  3 o  4 o protein HB aa (loose 3 o ) (breathing) < msUp to 1s

16. Levinthal & Landscapes Structure space 3 100 conformations Sequence space 20 100 sequences Figure from Englander & co-workers, Proc Natl Acad Sci 98 19104 (2001)

18. Why won’t it fold? Most common obstacles to a native fold: Aggregation Non-native disulfide bridge formation Isomerization of proline

20. Folding landscapes and the Levinthal par adox Flat landscape (Levinthal paradox) Tunnel landscape (discrete pathways) Realistic landscape (“folding funnel”)

21. Escherichia coli chaperonin (GroE)

22. Chaperonins / Heat Shock Proteins HSPs help proteins fold by preventing aggregation Recognize only unfolded proteins – Not specific – Recognizes exposed HB patches – Prevent aggregation of unfolded or misfolded proteins HSP70 – Assembly & disassembly of oligomers – Regulate translocation to ER HSP60 (GroEL) & HSP10 (GroES) – Work as a complex

23. Each subunit – Apical (  motif) Opening of chaperone to unfolded protein Flexible HB – Intermediate (  helices) Allow ATP and ADP diffusion Flexible hinges – Equatorial (  helices) ATP binding site Stabilizes double ring structure – Central cavity up to 90Å diam. 7 subunits in one ring 2 rings back to back GroEL

24. Cap to the GroEL Each subunit –  sheet –  hairpin (roof) – Mobile loop (int w/ GroEL) 7 subunits in functional molecule GroES

25. GroEL+ GroES work together GroEL makes up a cylinder – Each side has 7 identical subunits – Each side can accommodate one unfolded protein 1 GroES binds to one side of GroEL at a time – Allosteric inhibition at other site One side of cylinder is actively folding protein at a time

26. 1.GroEL/ATP complex at side A 2.Bind GroES on this side 7 ATP  7 ADP this side has a wider cavity but closed top other side has smaller cavity and open top 3.Side B ring binds unfolded protein GroES falls off of side A ADP falls off of side A 4.Side B ring binds 7 ATPs 5.GroES binds GroEL/ATP 7 ATP  7 ADP protein folding occurs 6.Side A ring binds 7 ATPs protein folding occurs 7 ATP  7 ADP (side A) 7 ADP & GroES (side B) falls off 7.Side A ring binds next unfolded protein

27. Switch side of ATP binding each time Switch side of GroES binding for each folding rxn Switch side of protein docking for each folding rxn Fink, Chaperone Mediated Folding, Physiological Reviews, 1999 Mechanism of Chaperonin Function

29. EC 3.6.1.1 EC 2.6.1.2 EC 1.1.1.27

30. EC 6.3.1.2 EC 5.1.1.1 EC 4.1.1.1 www.chem.qmul.ac.uk/iubmb/enzyme/

32. Analóg tranzitného stavu v aktívnom mieste adenozíndeaminázy

37. How DNA Sequence Is Determined? Polyacrylamide Gel Electrophoresis ATC 32 P AT 32 P A T A G C T A C G ATCG 32 P ATCGA 32 P ATCGAT 32 P ATCGATC 32 P ATCGATCG 32 P ATCGATCGA 32 P ATCGATCGAT 32 P DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis But these bands can’t tell us the identity of the terminal nucleotides If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence Juang RH (2004) BCbasics

38. Sanger's Method: Maxam-Gilbert's Method: How to Obtain DNA Fragments 32 P ATCGATCG 32 P ATCG AT ATCGAT ATCG TAGCTAGCTA ATCGA 32 P Specific Reaction to G ATCG STOP A Terminated Keep on going Biosynthetic method Chemical method Template or Non-radioactive (invisible) 32 P A,T,C,G A Analogue Destroy → Cleavage ATCGATCGAT Producing various fragments Juang RH (2004) BCbasics

39. Phosphodiester bond P R P R P R P R P R P R OH 5’ 3’ 1 5’ A 1 2 3 4 5 6 A PO 4 2- H 3’ 5’ 2 H dideoxynuceotide Terminated ddNTP Sanger’s Method: How Terminated Normal Linking Can not react Juang RH (2004) BCbasics

40. Structure of the reversible terminator 3'-O-azidomethyl 2'-deoxythymidine 5'- triphosphate labeled with a removable fluorophore. Source: Bentley et al. (2008). Nature 456: 53–59.