NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Expression of secretory/periplasmic proteins and “soluble” domains of membrane proteins

Frank R. CollartMidwest Center for Structural Genomics

Biosciences Division

Argonne National Laboratory

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Goal

Goal: Evaluate high throughput methods for the cloning, expression and solubility analysis of secretory/periplasmic proteins and soluble domains of membrane proteins.

•Evaluate success rate from expression to crystal structure

•Identify obstacles or components that require improvement

•Determine suitability for incorporation into the MCSG pipeline

Target groupsHT

methods/screens

Oligoprogram

Helical membrane proteins

Secretory and periplasmic

proteins

Informatics and expression tools

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Experimental Approach

Bacillus subtilis – domain set SignalP used to identify signal sequences

First 70 amino acids HMM and NN options

TMHMM for membranes spanning segments Filtered for size and methionine content 205 targets. First pass (pMCSG7 vector) Standard HT methods used for cytoplasmic

targets

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Soluble clone descriptions

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Summary of expression experiments

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8

% s

olu

ble

Summary of solubility outcomes for cloning and expression of secretory proteins and domains of helical membrane proteins.

Category #of

targets # soluble % soluble

(category)

Secretory/N-terminal anchor 74 36 49

1 membrane spanning helix 39 15 38

2 membrane spanning helices 16 3 19

3 membrane spanning helices 20 5 25

4 membrane spanning helices 12 3 25

5 membrane spanning helices 11 2 18

6-12 membrane spanning helices 33 10 30

Total 205 79 39

Tota

l

5+

MS

H

4 M

SH

3 M

SH

2 M

SH

1 M

SH

N-te

rm S

/A

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Solubility distribution based on target class and topology

0

10

20

30

40

50

60

70

80%

of

tota

llow solubility

medium solubility

high solubility

Classification

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Results Summary

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

APC1972 model

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

0 200 400 600 800

APC2031*

APC2032*

APC1942

APC1945

APC1940*

APC1941*

APC2059

APC2077

APC2083

APC1924

APC2025

APC2063 Soluble domains

Topology cartoons for targets with 2-MSH

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Soluble domains – multiple TMH6+-MSH          

APC1910

19 11 4 6 12 11 8

19 4 4

139 4 6

175

APC1932

113 4 6

81 4

19 30 14 19

APC1917

134 9 14 132 19 19

1 19 27 6 20 35

APC1979

131 20 12 37 31

19 14 25 19

APC1918

135 9 14 131 19 19

1 19 27 6

20 35

APC1988

39 4 3

39 3 4

155

20 4 6

12 11 9

APC1926

140 24 3

24

28 12 6

APC2003130 19 34

29 12 8

APC1930

144 11 6

341 6 6

3 23 25 4

24 16

APC2011

14 9

47 44 230

12 6 6 18

3-MSH     4 and 5-MSH    

APC190220

1603 APC2012

138 20

4 3

APC1938129 19

20 15

APC2018 4 144

210

APC2064124 14

6 167

APC1901146 27

147 7

APC206812

1625 APC1946

157 14 30

17 6 4

APC2079126 20

31 APC19688 6 161

3 14

Key:

# of membrane spanning helices

Target ID inside outside

inside outside (target)

membrane

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Eukaryotic proteins - Domain scanning approach

1500 1600 18001700100

Informatic analysis

HT screen

Automated design

component

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Domain primer tool

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Domain permutations expressed in E. coli

Tag detection-Expression

Tag detection-Solubility

Target plate map

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Human endothelial cell protein MRSL66

660 amino acids

Domain 2- putativeGlycosyltransferase

Domain 1Membrane anchor

IKLILDTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYGSVCSIKLILDTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYIKLILDTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSIKLILDTRRAISEANEDPEP VEAECHWADTELNRRRIKLILDTRRAISEANEDPEP VEAECHWADTE

---------DTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYGSVCS---------DTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGY---------DTRRAISEANEDPEP VEAECHWADTELNRRRRRFCS---------DTRRAISEANEDPEP VEAECHWADTELNRRR---------DTRRAISEANEDPEP VEAECHWADTE

------------------- ISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYGSVCS

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Solubility analysis of MRSL66 domain permutations

-IKLILDTRRAISEANEDPEP Set 1- DTRRAISEANEDPEP Set 2 -ISEANEDPEP Set 3

Domain permutations

Set 1 Set 2 Set 3

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Summary

Secretory/periplasmic proteins and low complexity helical membrane protein are targets for SG pipelines.

Optimization Improved methods for prediction of signal sequences and

the boundaries of the soluble domains of helical membrane proteins (i.e. specifically determine the N- and C-terminus of the domains)

Tool development Need to evaluate alternative strategies such as

periplasmic expression systems or the incorporation of fusion tags to enhance.

NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Acknowledgements

Andrzej Joachimiak PI, MCSG

Shiu Moy Cloning/expression

Denise HolzleCloning/expression

Natalie Maltsev CMT/ANL

Gong Xi Yu CMT/ANL

Lynda Dieckman BIO/ANL

Diane Rodi BIO/ANL

The MCSG team