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Systematic Identification of Protein Domains for Structure Determination. Ming Luo, Ph.D. University of Alabama at Birmingham March 29, 2004 NIH. Current Progress on C. elegans Proteins. * Unique ORFs, each expressed and purified multiple times. Markley. Domain Identification. Methods - PowerPoint PPT Presentation
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Systematic Identification of Protein Domains for Structure
Determination
Ming Luo, Ph.D.
University of Alabama at Birmingham
March 29, 2004
NIH
Current Progress on C. elegans Proteins
Nov, 03 Mar, 04 INCREASECLONED 4762 7326 2564
EXPRESSED 2293 3128 835SOLUBLE 368 503 135
PURIFIED 152 189 37CRYSTALLIZED 58 65 7
X-RAY DATA 14 18 4STRUCTURE 9 11 2 + 1
Protein expressed (1 mL)
0
700
1400
2100
2800
3500
Oct-00
Apr-01
Oct-01
Apr-02
Oct-02
Apr-03
Oct-03
Apr-04
Soluble confirmed (1 L)
0
125
250
375
500
625
Oct-00
Apr-01
Oct-01
Apr-02
Oct-02
Apr-03
Oct-03
Apr-04
Purified (6 L)
0
40
80
120
160
200
Oct-00
Apr-01
Oct-01
Apr-02
Oct-02
Apr-03
Oct-03
Apr-04
Clone & expression
0
1500
3000
4500
6000
7500
Oct-00
Apr-01
Oct-01
Apr-02
Oct-02
Apr-03
Oct-03
Apr-04
Selected ORFsSelected ORFs ClonedCloned Expressed Expressed Soluble (1 L)Soluble (1 L) Purified * (6 L)Purified * (6 L)
4/7/2003 14,440 2,342 1,369 268 110
3/7/2004 15,556 7,326 3,218 503 189
* Unique ORFs, each expressed and purified multiple times.
Domain Identification
Methods
1. Conserved Sequence
(e.g. Pfam)
2. Spontaneous Degradation
3. Proteolysis
4. Functional Data
Markley
Predict Domains by Sequence
Program used: SMART (http://smart.embl-heidelberg.de/)Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864
Letunic et al. (2002) Nucleic Acids Res 30, 242-244
2-H91 35629 286
1 34655 320
11-D11
1 647323 475
28-C5
1 31325 273
11-E3
1 320151 304
11-D5
1 50041 436
20-D7
278 283
Four expressedOne solubleNone purified
Spontaneous Degradation
1F11 76F6 3D2
Purified protein samples were stored at 4°C over one month.
Mass Spectrometry
31.3141193404-Nov-200306:43:10
5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000 32500 35000 37500 40000 42500 45000 47500 50000mass0
100
%
18H1 88 (3.002) M1 [Ev-64188,It10] (Gs,0.750,627:1371,1.00,L50,R50); Sm (Mn, 2x3.00); Cm (87:90-54:85) TOF MS ES+ 1.11e325440
12720
8480
6360 95392471419079 22012
38955337503109627753 36454
454954367641164 47376
Eluted from Gel
18H1
Solution Specimen
3D2
30.9721527124-Sep-200315:28:43
5000 10000 15000 20000 25000 30000 35000 40000 45000 50000mass0
100
%
3D2_NATIVE 24 (0.824) M1 [Ev-77696,It12] (Gs,0.750,843:1558,1.00,L50,R50); Sm (Mn, 2x3.00); Cm (20:40-1:12) TOF MS ES+ 79016809
8984 12340
17969
33618
31214
287502467922412
19961
26953
48183
4482335938 4291637192 40156
46631
MS + AA Sequencing
MS19695
AA CodeSAIKD
140-309379
MS21279
AA CodeGSQSTSL
18-210261
76F6 3D2
Proteolysis
Trypsin Digestion
1. Trypsin:protein 1:200, 10 mM Tris, pH7.6, 37°C.
2. N-terminal Sequencing after transfer to PVDF
ELTSAEK---
3. Mass Spectrometry using solution mixture
1927717774
Result: 59-212
Min 0 5 10 15 20 60 MW
9H3
Functional Data
1D10
Predicted Signal Peptide parameters from Soren Brunak's SignalP server:
Signal peptide predicted:
HMM-cleavage prediction: MPKLPLLLSFPLLFFASFAYA--(22)DEDFVT
ANN-cleavage prediction: MPKLPLLLSFPLLFFASFAYA--(22)DEDFVT
79D4
SUMMARY
Total# of ORFs 14 23 8 6 51
Domain ID by Degradation Proteolysis Functional Sequence
11 11 8 7 37Expressed 10 7 5 4 26
Soluble 10 7 5 1 23Purified 7 1 3 0 11
Xtal 5/(1 NMR) 1 2 0 8Structure 3 0 2 0 5
SUMMARY OF DOMAIN IDENTIFICATION
CONCLUSIONS
Smaller structural domains are most suitable for HTP structure determination.
Domains experimentally identified from folded proteins are most reliable.
Spontaneously occurring or limited proteolysis, followed by N-terminal sequencing and mass spectrometry, are most efficient approaches.
Our Team
TARGET SCREEN AND AUTOMATION* Chi-Hao Luan , Team Leader Wen Ying Huang ShiHong Qiu Zhuhua Cao Rita Gray Qiao Shang
PROTEIN PRODUCTION* Robert Bunzel , Team Leader Danlin Luo, Jennifer Zhou Alireza Arabshahi Elizaveta Karpova Annette McKinstry
CRYSTALLIZATION* Larry DeLucas , Team Leader Songlin Li Youhong Zhang
X-RAY CRYSTALLOGRAPHY* Songlin Li , Team Leader Jindrich Symersky Norbert Schormann Guangda Lin Shanyun Lu
BIOINFORMATICS* Mike Carson , Team Leader David Johnson Jun Tsao
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