Zakiya Qualls

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 Side view of hexamer Middle domain (red) Channel Middle domain (red) N-terminal domain (green) ATP (yellow) NBD-1 ATP NBD-2 Channel Model of E. coli ClpB based on T. thermophilus structure. Structure: Lee et al., Cell, 2003 Hexamer model: Diemand & Lupas, J. Struct. Biol., 2006 • 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.

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. • Construct N-domain and M-domain mutants. • Purify active ClpB mutant proteins. • Examine the unfolding capabilities of the ClpB mutant proteins compared with wild-type ClpB. • Examine GFP disaggregation by ClpB mutant proteins.

6. ClpB N-domain and M-domain Mutants 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. N-1 M-1 M-3 M-2

7. Methods Mutant Construction: • Selected sites for mutagenesis • Designed primers • Mutagenesis of ClpB gene (QuickChange II Site-Directed Mutagenesis Kit) • Transformation of plasmids (DH5 cells) • 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 ClpB 1ul 2.5ul 7.5ul 2ul 8ul 15ul P S P S Induced with IPTG High Speed Uninduced Low Speed

8. 38 31 32 33 35 36 37 8 9 10 11 12 13 14 15 16 29 30 34 ClpB(M-1) 8 9 10 11 12 13 14 15 16 110 kDa 110 kDa 110 kDa 110 kDa 80 kDa 80 kDa 80 kDa 80 kDa 60 kDa 60 kDa 60 kDa 60 kDa 22 15 17 14 17 18 19 20 21 16 9 10 11 12 13 14 M.W. 15 16 16 17 18 19 20 21 22 23 50 kDa 50 kDa 50 kDa 50 kDa 40 kDa 40 kDa 40 kDa 40 kDa ClpB(M-3) ClpB(M-2) 16 15 17 18 19 20 21 22 23 24 11 12 13 14 15 16 17 18 ClpB(N-1) Protein Purification - Column Chromatography S200 Separation by size and shape Q-sepharose Separation by charge

9. Unfolding of tagged-GFP by ClpB Incubate with ClpB and nucleotide Measure decrease in fluorescence Native fluorescent tagged-GFP (Green Fluorescent Protein) Unfolded non-fluorescent GFP N-terminal domain mutants M-domain mutants No ClpB No ClpB N-2 M-2 WT M-1 N-1 M-3 WT • 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.

10. Dissagregation of GFP by ClpB Incubate with ClpB and nucleotide Heat-aggregated non-fluorescent GFP Measure increase in fluorescence Refolded GFP M-domain mutants N-terminal domain mutants N-1 WT WT N-2 M-2 M-1 M-3 No ClpB No 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

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

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