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Systematic Identification of Protein Domains for Structure Determination

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

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  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 * Unique ORFs, each expressed and purified multiple times.

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

  4. 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

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

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

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

  8. 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

  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

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