Post on 30-Dec-2015
description
The Importance of Non-conserved Regions in Protein Remodeling by the E. coli Molecular Chaperone,
ClpB
Zakiya Qualls
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.
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.
ClpBTop 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, 2003Hexamer model: Diemand & Lupas,
J. Struct. Biol., 2006
Middle domain(red) N-terminal
domain(green)
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.
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.
Methods Mutant Construction:
1. Selected sites for mutagenesis2. Designed primers3. 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 spin4. Column Chromatography
Cell lysate of ClpB M-3 mutant SDS-PAGE
Induced with IPTG Uninduced
P S P S1ul 2.5ul 7.5ul2ul 8ul 15ul
High Speed
Low Speed
ClpB
Protein Purification - Column Chromatography Q-sepharose
Separation by charge S200
Separation by size and shape
17 188 1512 1410 11 16139 2316 222119 20
ClpB(M-2)
M.W.
110 kDa80 kDa
60 kDa
50 kDa40 kDa
171512 1410 11 16139 14110 kDa80 kDa
60 kDa
50 kDa40 kDa
ClpB(M-3)
17 1815 16 222119 20
29 30 32 343331 35 36 37 38
ClpB(M-1)
110 kDa80 kDa
60 kDa
50 kDa40 kDa
8 9 10 11 12 13 14 15 16
17 1815 16 22 23 242119 2011 12 13 14 15 16 17 18
ClpB(N-1)
110 kDa80 kDa
60 kDa
50 kDa40 kDa
Unfolding of tagged-GFP by ClpB
Native fluorescent tagged-GFP(Green Fluorescent Protein)
Incubate with ClpB andnucleotide
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.
NoClpB
WT
M-2M-1M-3
0.75
0.8
0.85
0.9
0.95
1
1.05
0 10 20 30 40 50 60Time (min)
Fluorescence Intensity
(A.U.)
M-domain mutants
0.45
0.55
0.65
0.75
0.85
0.95
1.05
0 10 20 30 40 50 60
Time (min)
Fluorescence Intensity
(A.U.)
No ClpB
WTN-2
N-1
N-terminal domain mutants
Measure increase in fluorescence
Incubate with ClpBand nucleotide
Heat-aggregatednon-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
0
10
20
30
40
50
60
0 10 20 30 40 50 60
Time (min)
Fluorescence Intensity
(A.U.)
0
10
20
30
40
50
60
0 10 20 30 40 50 60
Time (min)
Fluorescence Intensity
(A.U.)
NoClpB
N-1WTN-2
No ClpB
WT
M-2
M-1M-3
M-domain mutants N-terminal domain mutants
Measure increase in fluorescence
Incubate with ClpB + DnaK chaperone system
and nucleotide
Heat-aggregatednon-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-2M-1
M-3
DnaK chaperone system alone
0
20
40
60
80
100
120
140
0 10 20 30 40 50 60
Time (min)
Fluorescence Intensity
(A.U.)
M-domain mutants
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.
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