1. Ming Luo, Ph.D. University of Alabama at Birmingham March 29, 2004 NIH
Systematic Identification of Protein Domains for Structure Determination
Ming Luo, Ph.D.
University of Alabama at Birmingham
March 29, 2004
NIH

2. Current Progress on C. elegans Proteins
Selected ORFs
Cloned
Expressed
Soluble (1 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.

3. Domain Identification
Markley
Methods
1. Conserved Sequence
(e.g. Pfam)
Spontaneous Degradation
3. Proteolysis
4. Functional Data

4. Predict Domains by Sequence
2-H9 1
356 29
286
346 55
320
11-D11
647
323
475
28-C5
313 25
273
11-E3
151
304
11-D5
500 41
436
20-D7
Program used: SMART (
Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, Letunic et al. (2002) Nucleic Acids Res 30,
Four expressed
One soluble
None purified

5. Spontaneous Degradation
1F11
76F6
3D2
Purified protein samples were stored at 4°C over one month.

6. Mass Spectrometry
Solution Specimen
3D2
Eluted from Gel
18H1

7. MS + AA Sequencing 76F6 3D2 21279 18-210 GSQSTSL 261 MS 19695 140-309
AA Code
SAIKD
379
21279
GSQSTSL
18-210
261
76F6
3D2

8. Proteolysis Trypsin Digestion Min 0 5 10 15 20 60 MW
9H3
Trypsin Digestion
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
19277
17774
Result:

9. 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

10. SUMMARY

11. 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.

12. Our Team