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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
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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 1. Conserved Sequence (e.g. Pfam) Spontaneous Degradation 3. Proteolysis 4. Functional Data
2-H9 356 1 29 286 11-D5 1 320 151 304 11-D11 346 1 55 320 11-E3 1 313 25 273 20-D7 1 500 41 436 278 283 28-C5 1 647 323 475 Predict Domains by Sequence Program used: SMART (http://smart.embl-heidelberg.de/) Schultz et al. (1998) Proc. Natl. Acad. Sci. USA95, 5857-5864Letunic et al. (2002) Nucleic Acids Res30, 242-244 • Four expressed • One soluble • None purified
1F11 76F6 3D2 Spontaneous Degradation Purified protein samples were stored at 4°C over one month.
Solution Specimen Eluted from Gel 3D2 18H1 Mass Spectrometry
MS 21279 AA Code GSQSTSL 18-210 261 MS 19695 AA Code SAIKD 140-309 379 MS + AA Sequencing 76F6 3D2
Min 0 5 10 15 20 60 MW 9H3 Proteolysis 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: 59-212
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
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.