Download - Cal State LA - Instructional Web Server

Computational studies of
intramolecular disulfide bonded
catenanes as a novel stabilizing
mechanism in thermophilic microbes
August 23, 2007
Daniel Park
Yeates lab, MBI, UCLA
SoCalBSI
Today
• Intracellular disulfide abundance in
thermophiles/hyperthermophiles
• P. aerophilum citrate synthase
• Searching for catenanes
• Results
Importance of studying thermophilic
enzymes
• Industrial applications
– Engineering heat-stable biomolecules
– Utilizing those found in nature
• Taq DNA polymerase for PCR
• Insight into protein folding mechanisms
– Evolution of thermostable proteins
Intracellular disulfide bond abundance
Mallick et al., 2002
PNAS 99, pp. 9679-9684
Presence of disulfide bonds within the
intracellular proteins of P. aerophilum
• Both lanes reduced
• Presense and absence of
iodoacetamide
• Large fraction of P.
aerophilum proteins
contain disulfide bonds
Boutz et al., 2007
JMB 368, pp. 1332-1344
Citrate synthase (PaCS) from P. aerophilum
Boutz et al., 2007
JMB 368, pp. 1332-1344
Catenane structure of PaCS
Boutz et al., 2007
JMB 368, pp. 1332-1344
Disulfide bonds: contribution to the
thermostability of PaCS
Boutz et al., 2007
JMB 368, pp. 1332-1344
Cysteine abundance at terminal regions
1.4
N-term (20 residues)
middle
C-term (20 residues)
Cys abundance (%)
1.2
1
0.8
0.6
0.4
0.2
0
P. aerophilum
(2605 proteins)
A. pernix (1700
proteins)
E. coli K-12 (4131 S. cerevisiae (5879
proteins)
proteins)
organism
Alignment of thermophilic citrate synthase
Approach
Possible catenanes by temperature
Cysteine abundance at terminal regions
2
1.8
Cys abundance (%)
1.6
N-term (20 residues)
middle
C-term (20 residues)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
P. islandicum
DSM 4184
A. pernix
M. thermophila
PT
organism
T. elongatus
D. psychrophila
LSv54
Clusters of orthologous groups (COG)
functional classifications
•
•
•
•
•
•
INFORMATION STORAGE AND PROCESSING
[J] Translation, ribosomal structure and biogenesis
[A] RNA processing and modification
[K] Transcription
[L] Replication, recombination and repair
[B] Chromatin structure and dynamics
•
•
CELLULAR PROCESSES AND SIGNALING
[D] Cell cycle control, cell division, chromosome
partitioning
[Y] Nuclear structure
[V] Defense mechanisms
[T] Signal transduction mechanisms
[M] Cell wall/membrane/envelope biogenesis
[N] Cell motility
[Z] Cytoskeleton
[W] Extracellular structures
[U] Intracellular trafficking, secretion, and vesicular
transport
[O] Posttranslational modification, protein turnover,
chaperones
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
METABOLISM
[C] Energy production and conversion
[G] Carbohydrate transport and metabolism
[E] Amino acid transport and metabolism
[F] Nucleotide transport and metabolism
[H] Coenzyme transport and metabolism
[I] Lipid transport and metabolism
[P] Inorganic ion transport and metabolism
[Q] Secondary metabolites biosynthesis, transport
and catabolism
•
•
•
POORLY CHARACTERIZED
[R] General function prediction only
[S] Function unknown
Possible microbial catenanes by function
Possible microbial catenanes by function
Possible thermophilic catenanes by function
Possible thermophilic catenanes further
classified by COGs (top 7)
Functional
classification
COG type
No. of
catenanes
[O] Posttranslational
modification, protein
turnover, chaperones
Peroxiredoxin
7
[C] Energy production
and conversion
Citrate synthase
5
[C] Energy production
and conversion
Anaerobic dehydrogenase, typically
selenocysteine-containing
5
[?]
Unclassified
5
[G] Carbohydrate
transport
Transketolase, N-terminal subunit
4
[EP] Amino acid and
inorganic ion transport
ABC-type dipeptide/oligopeptide/nickel
transport system
4
[E] Amino acid transport
3-dehydroquinate synthetase
4
Possible catenane among peroxiredoxin
homologs?
•
[O] COG0450 Peroxiredoxin (7)
Thermoanaerobacter tengcongensis MB4
Methanosaeta thermophila PT
Pyrobaculum islandicum DSM 4184
Pyrobaculum islandicum DSM 4184
Pyrobaculum calidifontis JCM 11548
Pyrobaculum arsenaticum DSM 13514
Methanocaldococcus jannaschii DSM 2661
•
20808569
116754713
119873344
119871684
126458809
145590729
15668917
[C] COG0372 Citrate synthase (5)
Pyrobaculum islandicum DSM 4184
Pyrobaculum calidifontis JCM 11548
Pyrobaculum arsenaticum DSM 13514
Pyrobaculum aerophilum str. IM2
Aeropyrum pernix K1
119873179
126459178
145592430
18312809
14601576
P. islandicum DSM 4184 peroxidase:
alignment with homologs
P. islandicum peroxidase homolog
P. islandicum peroxidase homolog
Future directions
• MD simulations of possible catenanes
• Determine structures of most likely catenanes
by X-ray crystallography
• Investigate correlation between psychrophilic
proteins and disulfide bonding
Acknowledgements
•
•
•
•
Todd Yeates
Neil King
Jason Forse
Brian O’Connor
•
•
•
•
Jamil Momand
Sandra Sharp
Wendie Johnston
Nancy Warter-Perez
• SoCalBSI program
• Ronnie Cheng
• Funded by NIH, NSF, EWD, DOE