Transcript Slide 1
Denaturácia a renaturácia RNázy A Nobelova cena z chémie v roku 1972 za práce o zvinovaní proteínov Anfinsen Experiment • Denaturation of ribonuclease A (4 disulfide bonds), with 8 M Urea containing bmercaptoethanol, leads to random coil and no activity 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. Anfinsen Experiment • Remove b-mercaptoethanol only, oxidation of the sulfhydryl group, then remove urea → scrambled protein, no activity • Further addition of trace amounts of b-mercaptoethanol converts the scrambled form into native form. • Conclusion: The native form of a protein has the thermodynamically most stable structure. Models of Protein Folding Framework model of protein folding C N Supported by experimental observation of rapid formation of secondary structure during protein folding process Framework model of protein folding C N Formation of individual secondary structure elements Framework model of protein folding N C Coalescence and rearrangement of individual secondary structure elements Nuclear condensation model C N Supported by protein engineering studies and various theoretical calculations Nuclear condensation model N C Formation of a nucleus of hydrophobic residues Nuclear condensation model N C Expansion of nucleus Steps of Folding < ms Up to 1s Unfolded bury core 2o Molten globule 3o 4o protein HB aa (loose 3o) (breathing) Levinthal & Landscapes • Structure space 3100 conformations • Sequence space 20100 sequences Figure from Englander & co-workers, Proc Natl Acad Sci 98 19104 (2001) Why won’t it fold? Most common obstacles to a native fold: • Aggregation • Non-native disulfide bridge formation • Isomerization of proline Folding landscapes and the Levinthal paradox Flat landscape (Levinthal paradox) Tunnel landscape (discrete pathways) Realistic landscape (“folding funnel”) Escherichia coli chaperonin (GroE) 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 GroEL • Each subunit – Apical (a/b motif) • Opening of chaperone to unfolded protein • Flexible • HB – Intermediate (a helices) • Allow ATP and ADP diffusion • Flexible hinges – Equatorial (a helices) • ATP binding site • Stabilizes double ring structure – Central cavity up to 90Å diam. • 7 subunits in one ring • 2 rings back to back GroES • Cap to the GroEL • Each subunit – b sheet – b hairpin (roof) – Mobile loop (int w/ GroEL) • 7 subunits in functional molecule 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 1. GroEL/ATP complex at side A 2. Bind GroES on this side 7 ATP7 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 ATP7 ADP protein folding occurs 6. Side A ring binds 7 ATPs protein folding occurs 7 ATP7 ADP (side A) 7 ADP & GroES (side B) falls off 7. Side A ring binds next unfolded protein Mechanism of Chaperonin Function • 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 EC 1.1.1.27 EC 2.6.1.2 EC 3.6.1.1 EC 6.3.1.2 EC 5.1.1.1 EC 4.1.1.1 www.chem.qmul.ac.uk/iubmb/enzyme/ Analóg tranzitného stavu v aktívnom mieste adenozíndeaminázy How DNA Sequence Is Determined? DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis 32P 32P 32P 32P 32P 32P 32P 32P ATCGATCGAT ATCGATCGA ATCGATCG ATCGATC ATCGAT ATCGA ATCG ATC 32P AT 32P A But these bands can’t tell us the identity of the terminal nucleotides Polyacrylamide Gel Electrophoresis T A G C G C T A If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence Juang RH (2004) BCbasics How to Obtain DNA Fragments Chemical method Maxam-Gilbert's Method: ATCGATCGAT 32P ATCGATCG 32P ATCG Specific Reaction to G ATCG TAGCTAGCTA Template Biosynthetic method 32P Keep on going ATCGA Analogue TAGCTAGCTA A A,T,C,G ATCGAT Non-radioactive (invisible) Destroy → Cleavage Sanger's Method: 32P AT or Producing various fragments STOP ATCG Terminated A 32P Destroy → Cleavage TAGCTAGCTA Juang RH (2004) BCbasics P 1 R 5’ P 1 2 ddNTP R 3’ P OH R 5’ 2P PO4 A R Phosphodiester P bond 2 R P 3’ H H R Normal Linking Sanger’s Method: How Terminated 5’ dideoxynuceotide Can not react A Terminated 3 4 5 6 3’ Juang RH (2004) BCbasics 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.