This document summarizes experiments to locate regions in the bacterial protein DnaK that are important for its chaperone activity. DnaK is a molecular chaperone found in E. coli and similar proteins exist across organisms. It aids protein folding and protects cells from stress. The structure of DnaK and a peptide-binding mutant are described. Methods are outlined to mutate plasmids, overproduce proteins, and purify DnaK using chromatography. Future plans involve measuring chaperone activity by monitoring refolding of denatured green fluorescent protein. The significance is that inhibiting chaperones may help cure diseases caused by protein misfolding such as cancer and cystic fibrosis.

1. Locating regions in Dnak
important for chaperone activity
Roxana Hernandez
7/27/12
DNA Molecular Laboratory, NCI,NIH

2. DnaK
• Source from Escherichia
coli
• DnaK/Hsp70 (70
kilodalton heat shock
proteins) with similar
structure exist in almost
all living organisms
• Important for protein
folding and to help
protect cells from stress
• Functions with co-
chaperones

3. Structure of DnaK
ATP
ATPase
domain
peptide
Substrate-
binding
domain
Peptide bound model - Stevens, S.Y. et al., (2003)
Protein Science
Nucleotide bound model - Sousa, M.C. and McKay, D.B (1998)
Biochemistry
N- and C-terminal combined model based on Hsc70 structure - Jiang, J. et
al., (2005) Molecular Cell

4. Structure of mutant
Peptide
(substrate)
ATP
DnaK 351-355

5. Goals
• Inhibiting molecular chaperones involved
in activating oncogenic proteins could
help cure cancer

6. Methods
Mutated plasmids transformed into cells
for growth using ‘QuickChange’ for Site-
directed mutagenesis
Collected DnaK 351 mutated plasmid for
sequencing
If mutation is correct then the plasmid is
used for protein overproduction

7. Methods
• Protein prep:
① Grow culture
(overproduction of
proteins)
② Lyse cells using a
French Press
③ Column
Chromatography
kDa
181.8
82.2
64.2
48.8
37.1
25.9
19.4
M P S P S U I
14.8

8. Column Chromatography
Q-Sepharose
Anion Exchange
S100
Size Exclusion
kDa
181.
8115.5
82.2
37.1
48.8
25.9
19.4
64.2
70
D L W M D M

9. Conclusions

10. Future Plans
Monitor disaggregation by
measuring the increase in
fluorescence
Native Green
Fluorescent Protein
(GFP)
Heat
denature
Incubate with chaperones
& ATP
Refolded
GFP
Heat-aggregated
non-fluorescent GFP

11. Significance
• Investigating the role of chaperones in
diseases of protein misfolding
• Inhibiting the chaperone involved could
help cure disease
– Ex: Cancer, Cystic Fibrosis, Sickle Cell, Type II
Diabetes..

12. Acknowledgements
• Sue Wickner
• Shannon Doyle
• Joel Hoskins
• Danielle Johnston
• Olivier Genest
• Colleen Berringer
DNA Molecular Laboratory, NCI,NIH