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 Selected ORFs ClonedExpressed Soluble (1 L) Purified * (6 L) 4/7/200314,4402,3421, /7/200415,5567,3263, * 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 ( Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, Letunic et al. (2002) Nucleic Acids Res 30, H D C E D D  Four expressed  One soluble  None purified

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

Mass Spectrometry Eluted from Gel 18H1 Solution Specimen 3D2

MS + AA Sequencing MS AA Code SAIKD MS AA Code GSQSTSL F63D2

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 Result: Min 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

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.

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