denaturácia a renaturácia rnázy a nobelova cena z chémie v roku 1972 za práce o zvinovaní...
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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-mercaptoethanol, 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 -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.
Models of Protein Folding
Framework model of protein folding
Supported by experimental observation of rapid formation
of secondary structure during protein folding process
N C
Framework model of protein folding
N
C
Formation of individual secondary structure elements
Framework model of protein folding
N
C
Coalescence and rearrangement of
individual secondary structure elements
Nuclear condensation model
N C
Supported by protein engineering studies
and various theoretical calculations
Nuclear condensation model
NC
Formation of a nucleus of hydrophobic residues
Nuclear condensation model
N
C
Expansion of nucleus
Steps of Folding
Unfolded bury core 2o Molten globule 3o 4o
protein HB aa (loose 3o) (breathing)
< ms Up to 1s
Levinthal & Landscapes
• Structure space3100 conformations
• Sequence space20100 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
• 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
• Cap to the GroEL
• Each subunit– sheet– hairpin (roof)– Mobile loop (int w/ GroEL)
• 7 subunits in functional molecule
GroES
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 A2. Bind GroES on this side
7 ATP7 ADP this side has a wider cavity but closed topother side has smaller cavity and open top
3. Side B ring binds unfolded proteinGroES falls off of side AADP falls off of side A
4. Side B ring binds 7 ATPs5. GroES binds GroEL/ATP
7 ATP7 ADP protein folding occurs
6. Side A ring binds 7 ATPsprotein folding occurs
7 ATP7 ADP (side A) 7 ADP & GroES (side B) falls off
7. Side A ring binds next unfolded protein
• 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
EC 3.6.1.1
EC 2.6.1.2
EC 1.1.1.27
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?
Polyacrylamide Gel Electrophoresis
ATC32PAT32PA32P
T A G C
T
A
CG
ATCG32P
ATCGA32P
ATCGAT32P
ATCGATC32P
ATCGATCG32PATCGATCGA32P
ATCGATCGAT32P
DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis
But these bands can’t tell usthe identity of the terminal nucleotides
If those band with the sameterminal nucleotide can begrouped, then it is possible to read the whole sequence
Juang RH (2004) BCbasics
Sanger's Method:
Maxam-Gilbert's Method:
How to Obtain DNA Fragments
32P32P ATCGATCG
32P ATCG
AT
ATCGAT
ATCG
TAGCTAGCTA
TAGCTAGCTA
TAGCTAGCTA
ATCGA
32P
Specific Reaction to G
ATCG
STOP
A
Terminated
Keep on goingBiosynthetic method
Chemical method
Template
or
Non-radioactive(invisible)
32P A,T,C,G A
Analogue
Destroy → Cleavage
Destroy → Cleavage
ATCGATCGAT
Producing various fragments
Juang RH (2004) BCbasics
Phosphodiesterbond
P R P R P R P R P R P R
OH
5’
3’
1
3’
5’
A
1
2
3
4
5
6
APO4
2-
H3’
5’
2
Hdideoxynuceotide
TerminatedTerminatedddNTP
Sanger’s M
ethod: How
Term
inated
Normal
Linking
Can not reactJuang 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.
