1. 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 ClonedExpressed Soluble (1 L) Purified * (6 L) 4/7/200314,4402,3421,369268110 3/7/200415,5567,3263,218503189 * Unique ORFs, each expressed and purified multiple times.

3. Domain Identification Methods 1.Conserved Sequence (e.g. Pfam) 2.Spontaneous Degradation 3.Proteolysis 4.Functional Data Markley

4. 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 -2445857-5864242 -244 2-H9 1 356 29286 1 346 55320 11-D11 1647 323475 28-C5 1313 25273 11-E3 1320 151304 11-D5 1500 41436 20-D7 278 283  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 Eluted from Gel 18H1 Solution Specimen 3D2

7. MS + AA Sequencing MS 19695 AA Code SAIKD 140-309 379 MS 21279 AA Code GSQSTSL 18-210 261 76F63D2

8. Proteolysis Trypsin Digestion 1.Trypsin:protein1:200, 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: 59-212 Min 0 5 10 15 20 60 MW 9H3

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 4 Smaller structural domains are most suitable for HTP structure determination. 4 Domains experimentally identified from folded proteins are most reliable. 4 Spontaneously occurring or limited proteolysis, followed by N-terminal sequencing and mass spectrometry, are most efficient approaches.

12. Our Team