Recommended
More Related Content
Similar to Locating regions of Escherichia coli Dnak important for molecular chaperone activity
Similar to Locating regions of Escherichia coli Dnak important for molecular chaperone activity (18)
Recently uploaded
Recently uploaded (14)
Locating regions of Escherichia coli Dnak important for molecular chaperone activity
- 1. Chosen because: § Surface exposed § Not previously identified as binding co-chaperone Mutagenesis of DnaK: § Mutagenesis of DnaK gene using QuikChange Site-Directed Mutagenesis Kit § Sequence DnaK gene to identify correct substitution Locating regions of Escherichia coli DnaK important for molecular chaperone activity Roxana Hernandez, Shannon Doyle, Danielle Johnston, and Sue Wickner DNA Molecular Biology Section, LMB, NCI, NIH 1. Background 6. ATPase activity of DnaK(351-355) 8. Future Plans 3. Mutagenesis of DnaK ATPase domain 4. Overproduction of DnaK mutant protein § Grow culture to OD600=0.8 (1) § Induce by heat (42°C) for 2 hours (2) § Collect cells and lyse with french press § Remove cell debris: supernatant (3) and pellet(4) § Clarify cell extract: supernatant (5) and pellet (6) 5. Purification of DnaK(351-355) (1) (2) (3) (4) (5) (6) DnaK: 70 kDa Q-Sepharose: Anion Exchange S100: Size Exclusion Separates proteins by charge Separates proteins by size and shape DnaK Q18 S100 Fractions Q18 DnaK NaCl § Molecular chaperones help proteins achieve their active/native conformation § One chaperone, Hsp70, uses chemical energy from ATP hydrolysis to fold and reactivate proteins § DnaK consists of 2 domains § ATPase Domain: § Binds and hydrolyzes ATP § Substrate Binding Domain: § Substrate binds when ATP is present and binding is stabilized by ADP § Substrate is released upon nucleotide exchange 2. Goal § DnaK functions with two co-chaperones § DnaJ (Hsp40) – targets substrates to Hsp70 and stimulates ATP hydrolysis by Hsp70 § GrpE (NEF) – promotes the exchange of nucleotide by Hsp70 § The Q18 fraction from the Q-Sepharose column was loaded onto an S100 column for further purification. Inactive substrate 90 JE K JE K ATP ATP Collaboration of Hsp90Ec with the DnaK system to promote remodeling Fully remodeled substrate Partial remodeling by the DnaK system J E K 90 Hsp90EcDnaK chaperone system ATPase domain ATP Substrate- binding domain Peptide (substrate ) 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 Model of DnaK Structure S M V Q K K V A E F F G K CCA ATG GTT CAG AAG AAA GTT GCT GAG TTC TTT GGT AAA CCA ATG GTT CAG GCG GCA GTT GGT GCG TTC TTT GGT AAA A A G A Amino Acids Nucleotides DnaK wild type sequence DnaK mutant sequence Substituted nucleotides and mutated amino acids A. ATPase Standard Curve OD620nm Inorganic phosphate (nmol) 0.00 0.04 0.08 0.12 0.16 0 0.5 1 DnaK wild-type DnaK 351-355 Mutant Actual values 7. Conclusions § The DnaK mutant can be overexpressed and is soluble § DnaK(351-355) was separated from other cellular proteins § ATPase activity corresponds with DnaK protein concentration § DnaK(351-355) has higher ATPase activity than wild-type DnaK 0.00 0.05 0.10 0.15 0.20 0.25 WT Mutant ATPhydrolysis nmolmin-1DnaK-1 B. ATPase activity of DnaK B. ATPase activities of DnaK(351- 355) column fractions 0 0.2 0.4 0.6 0.8 1 0.0 1.0 2.0 3.0 4.0 5.0 13 14 15 16 17 RateofATPhydrolysis(nmolmin-1) ProteinConcentration(mg/ml) S100 fraction number A. Methods 1. Bradford Assay Protein Concentration: 1. ATP Hydrolysis by DnaK ATPase Activity: Incubate reaction with BioMol Reagent Incubate with DnaK for 20 min ATP ADP + Pi 2. Detection of Pi Read absorbance At 620 nm Malachite Green Malachite Green Pi Does ATPase activity correspond with purified DnaK? How does the ATPase activity of DnaK(351-355) compare to wild-type DnaK activity? II. Clp/Hsp100s – dissolve protein aggregates § Fraction 15 has the highest ATPase activity correlating well with a high DnaK(351-355) concentration. § The Dnak(351-355) mutant has higher ATPase activity than DnaK wild-type § Compare DnaK(351-355) to wild-type DnaK in chaperone activity assays Introduction Hsp70/DnaK § The DnaK system collaborates with other chaperones I. Hsp90 – remodel substrates and modulate protein activity § Some Hsp70-Hsp90 substrates are known oncogenic proteins § Inhibiting the action of Hsp70 or Hsp90 could be a target for cancer therapies Collaboration with other chaperones § Hsp70 is found in almost every organism. In E. coli it is called DnaK. § To identify regions of DnaK that are important for chaperone activity Rate of hydrolysis Protein Concentration 40 NEF 70 Hsp70 chaperone system Non-native and inactive protein Native/active proteins 351-355 ATP Peptide (substrate)