STRUCTURAL PROTEOMICS

1

• STRUCTURAL PROTEOMICS
• Structural biology on a genome-wide scale
• Cheryl Arrowsmith
• Ontario Cancer Institute and
• Department of Medical Biophysics,
• University of Toronto

2
Cloning and Test Expression
ligate
transform
clones
PCR x96
24 x 3ml LB Kan, Amp 37C, Induce at OD600 Grow
O/N 15C or 20C
X 96
Kan, Amp
X 96
300 ul
300 ul
X 96
X 96
supernatant
Spin, Dissolve pellet in SDS
Spin, Freeze, Lyse with BugBusterTM Spin again
SDS PAGE
3
The NMR Pipeline
18 at a time in batch mode
Steady state rate 18 samples evaluated per week
4
Feasibility Results for MT Proteins
Christendat et al., 2000
gt 20 kDa
lt 20 kDa
cloned
expressed
soluble
purified
Crystals or good/promising HSQC
Well-diffracting crystals or good/promising HSQC
0
50
100
150
200
250
Number of Proteins
5
Extension of our initial feasibility study
6
Cloned
E. coli
Expressed
Soluble
yeast
T. maritima
Yee et al, PNAS, 2002
l phage
Myxoma virus
M. th.
7
Yee et al, PNAS, 2002
15N-HSQC Results for Soluble Proteins
PPT
PROMISING
POOR
UNFOLDED
GOOD
M. th.
yeast
E. coli
T. maritima
Myxoma virus
l phage
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Orthologues
68 Escherichia coli
68 Thermotoga maritima
Topt 80 C
Topt 37 C
1,860,725 bp
4,639,221 bp
1, 877 ORFs
4, 288 ORFs
Expressed soluble
62
48
Concentratable to gt 2mg/ml
50
44
9 Proteins could not be purified from either
species
35
15
9
9
Total Crystals (30)
T. maritima
E. coli
11
3
13
Total Good/Promising NMR spectra (14)
T. maritima
E. coli
2
4
4
10
NMR Crystallography complementary!
24 small proteins for which both crystal trials
and NMR data collected
Good/promising HSQC
crystals
3
10
6
Of 32 proteins that gave poor HSQCs 7 have
crystallized
11
150 M.th. Clones in Cell-Free Protein Expression
System
Total Protein
Soluble Protein
Clone
Shirouzu Yokoyama, RIKEN
12
Expressed Protein in Cell-Free System
E. Coli Expression
Not Soluble
Soluble
Total Protein
Soluble Protein
Clone
Shirouzu Yokoyama, RIKEN
13
Data storage and Mining Defined Vocabulary
Property Vocabulary Expression level 0-5 (no
expression high expression) Solubility (test
expression) 0-5 (insoluble highly
soluble) Concentratability 0-5 (or
mg/ml) Crystal trials clear amorphous
ppt granular ppt crystal Initial HSQC NMR
good promising poor
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Ontario Cancer Institute
Adelinda Yee (NMR) Brian Li Anthony
Semesi Cheng-Song Liu
Aled Edwards Limin Chen(Myxoma, l phage)

Alexei Savchenko (T.m. E.coli) Tania Skarina
Lena Evdokimova Marina Pavlova Nancy Ouellette
Dinesh Christendat (M.th.) Diane Bona J.
Gu Steven Beasley Micha Pennycooke Linda Wu
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Collaborators
S. Yokoyama (RIKEN) Mikako Shirouzu
Northeast Structural Genomics Consortium
Midwest Structural Genomics Consortium