1. The Importance of Non-conserved Regions in Protein Remodeling by the E. coli Molecular Chaperone, ClpB Zakiya Qualls

2. Background Molecular chaperones are a family of proteins that aid in the prevention of protein misfolding and aggregation. Protein aggregation often occurs following extreme environmental stress, for example heat stress, which can lead to the loss of protein activity and cell death. Protein aggregates, called amyloids are involved in diseases, including neurological disorders such as Alzheimer’s, Huntington’s, and Parkinson’s.

3. ClpB ClpB Is a molecular chaperone that is required for growth at high temperatures (thermotolerance). It belongs to the AAA+ (ATPases associated with various cellular activities) superfamily of ATPases. Both in vivo and in vitro, ClpB is required for protein disaggregation and reactivation. ClpB works with the DnaK chaperone system to disaggregate proteins. Yeast Hsp104, plant Hsp101, and mitochondrial Hsp78 are the homologs of ClpB.

4. ClpB Top view of hexamer ATP (yellow) Channel Middle domain (red) ATP Channel NBD-1 NBD-2 Side view of hexamer ClpB is a hexamer containing a central channel involved in protein unfolding and translocation. Each monomer is comprised of an N-domain and two nucleotide- binding domains (NBD) as well as a unique coiled-coil middle (M) domain. Model of E. coli ClpB based on T. thermophilus structure. Structure: Lee et al., Cell, 2003 Hexamer model: Diemand & Lupas, J. Struct. Biol., 2006 Middle domain (red) N-terminal domain (green)

5. Goals Question: What are the roles of the N-terminal domain and the M-domain in ClpB chaperone activity? To determine if mutations in the N-terminal domain and the M-domain of ClpB affect its protein remodeling abilities. 1.Construct N-domain and M-domain mutants. 2.Purify active ClpB mutant proteins. 3.Examine the unfolding capabilities of the ClpB mutant proteins compared with wild-type ClpB. 4.Examine GFP disaggregation by ClpB mutant proteins.

6. ClpB N-domain and M-domain Mutants M-2 M-3 M-1 N-1 N-2 The N- and M-domains have low sequence homology among species. An N-terminal deletion alters the function of ClpB both in vivo and in vitro. The M-domain is unique to ClpB and its homologs and required for chaperone activity.

7. Methods Mutant Construction: 1.Selected sites for mutagenesis 2.Designed primers 3.Mutagenesis of ClpB gene (QuickChange II Site-Directed Mutagenesis Kit) 4.Transformation of plasmids (DH5  cells) 5.Plasmid Prep (QIA prep Spin Miniprep Kit) 6. Confirmed mutations by sequencing Protein Preparation: 1. Transformation of mutant plasmid into BL21 cells) 2. Growth and induction 3. Low speed & high speed spin 4. Column Chromatography Cell lysate of ClpB M-3 mutant SDS-PAGE Induced with IPTG Uninduced P S 1ul 2.5ul 7.5ul 2ul 8ul 15ul High Speed Low Speed ClpB

8. Protein Purification - Column Chromatography Q-sepharose Separation by charge S200 Separation by size and shape 1718 8151214101116139 231622211920 ClpB(M-2) M.W. 110 kDa 80 kDa 60 kDa 50 kDa 40 kDa 17 15 12141011 16 139 14 110 kDa 80 kDa 60 kDa 50 kDa 40 kDa ClpB(M-3) 1718 1516 22 211920 2930 32 34 3331353637 38 ClpB(M-1) 110 kDa 80 kDa 60 kDa 50 kDa 40 kDa 8 9 10 11 12 13 14 15 16 1718 15 16 22 23 24211920 11 12 13 14 15 16 17 18 ClpB(N-1) 110 kDa 80 kDa 60 kDa 50 kDa 40 kDa

9. Unfolding of tagged-GFP by ClpB Native fluorescent tagged-GFP (Green Fluorescent Protein) Incubate with ClpB and nucleotide Unfolded non-fluorescent GFP Measure decrease in fluorescence The M-domain mutants are not significantly different than wild-type ClpB. The N-terminal domain mutants N-1 and N-2 are defective compared to wild-type ClpB. No ClpB WT M-2 M-1 M-3 M-domain mutants No ClpB WT N-2 N-1 N-terminal domain mutants

10. Measure increase in fluorescence Incubate with ClpB and nucleotide Heat-aggregated non-fluorescent GFP Refolded GFP Dissagregation of GFP by ClpB The three M-domain mutants are defective in protein disaggregation compared to wild-type ClpB. N-1 and N-2 have similar disaggregation activity compared to wild-type ClpB No ClpB N-1 WT N-2 No ClpB WT M-2 M-1 M-3 M-domain mutants N-terminal domain mutants

11. Measure increase in fluorescence Incubate with ClpB + DnaK chaperone system and nucleotide Heat-aggregated non-fluorescent GFP Refolded GFP Disaggregation of GFP by ClpB with DnaK Chaperone System The M-domain mutants are defective compared to wild-type ClpB in disaggregation activity with the DnaK chaperone system. No Chaperone WT M-2 M-1 M-3 DnaK chaperone system alone M-domain mutants

12. Conclusions ClpB mutants in the N -terminal domain have decreased protein unfolding activity compared to wild-type, but have disaggregation activity similar to wild-type in the absence of the DnaK chaperone system. ClpB M-domain mutants possess protein unfolding activity similar to wild-type, but have reduced disaggregation activity alone and in the presence of the DnaK system. The M-domain may be important for protein disaggregation by ClpB in the presence and absence of the DnaK chaperone system. This and further research will help understand how molecular chaperones interact with other proteins and how they may be vital in fighting several neurological disorders.

13. Acknowledgements Dr. Sue Wickner Shannon Doyle Danielle Johnston Jodi Camberg Joel Hoskins Marika Miot Olivier Genest NIH Summer Internship Program in Biomedical Research The Howard University COR Honors Research Program