1. Protein Properties Function, structure Residue features Targeting Post-trans modifications BIO520 BioinformaticsJim Lund Reading: Chapter 11.1-3, 11.7, 12.1, Ch 14

2. Protein function, structure Homology to known proteins through protein search and alignment Functional domains –NCBI CDD (conserved domain database) –Interpro (PROSITE, Pfam, SMART, etc.)

3. Simple Analyses Composition (#’s of each aa) Isoelectric point Extinction co-efficient Peptide cleavage Repetitive regions ExPASy, BCM Search Launcher, other sites.

4. CDD domains in Insulin-Like Growth Factor Receptor

5. Composition (Peptide) COMPOSITION of: (Human glyceraldehyde-3- phosphate dehydrogenase) *.pep Check: 5354 from: 1 to: 334 October 6, 1998 13:18 ***** A: 33 C: 3 D: 23 E: 16 F: 15 G: 34 H: 10 I: 21 K: 27 L: 20 M: 9 N: 13 P: 11 Q: 4 R: 10 S: 20 T: 21 V: 32 W: 3 Y: 9 Other: 0 Total: 334

6. Isoelectric Point(pI) Number of Hydrogen Ions Bound ---------------------------------------------------------- Net pH Arg Lys His Tyr Cys Glu Asp NH2 COOH Total Charge.. 1.00 10.00 27.00 10.00 9.00 3.00 15.99 22.97 1.00 1.00 99.96 47.96 1.50 10.00 27.00 10.00 9.00 3.00 15.97 22.91 1.00 0.99 99.87 47.87 2.00 10.00 27.00 10.00 9.00 3.00 15.91 22.72 1.00 0.97 99.60 47.60 2.50 10.00 27.00 10.00 9.00 3.00 15.72 22.14 1.00 0.92 98.78 46.78 3.00 10.00 27.00 10.00 9.00 3.00 15.15 20.48 1.00 0.78 96.41 44.41 3.50 10.00 27.00 9.99 9.00 3.00 13.58 16.56 1.00 0.53 90.67 38.67 4.00 10.00 27.00 9.97 9.00 3.00 10.24 10.31 1.00 0.27 80.79 28.79 4.50 10.00 27.00 9.90 9.00 3.00 5.76 4.70 1.00 0.10 70.47 18.47 5.00 10.00 27.00 9.69 9.00 3.00 2.42 1.73 1.00 0.04 63.87 11.87 5.50 10.00 27.00 9.09 9.00 3.00 0.85 0.58 1.00 0.01 60.52 8.52 6.00 10.00 27.00 7.60 9.00 2.99 0.28 0.19 1.00 0.00 58.05 6.05 6.50 10.00 27.00 5.00 9.00 2.95 0.09 0.06 0.99 0.00 55.09 3.09 7.00 10.00 27.00 2.40 9.00 2.86 0.03 0.02 0.97 0.00 52.27 0.27 7.50 10.00 26.99 0.91 9.00 2.59 0.01 0.01 0.92 0.00 50.42 -1.58 8.00 10.00 26.96 0.31 8.99 2.00 0.00 0.00 0.78 0.00 49.04 -2.96 8.50 10.00 26.86 0.10 8.97 1.16 0.00 0.00 0.53 0.00 47.63 -4.37 9.00 10.00 26.57 0.03 8.90 0.50 0.00 0.00 0.27 0.00 46.26 -5.74 9.50 9.99 25.68 0.01 8.69 0.18 0.00 0.00 0.10 0.00 44.66 -7.34 10.00 9.97 23.23 0.00 8.09 0.06 0.00 0.00 0.04 0.00 41.39 -10.61 10.50 9.90 17.85 0.00 6.64 0.02 0.00 0.00 0.01 0.00 34.42 -17.58 11.00 9.69 10.30 0.00 4.24 0.01 0.00 0.00 0.00 0.00 24.24 -27.76 11.50 9.09 4.41 0.00 1.98 0.00 0.00 0.00 0.00 0.00 15.48 -36.52

7. Extinction co-efficient 276278279280282.815.810.819.815.810.807.796.793.769.766 S-S SH 1  g/ml Used for determining protein concentration. Based on number C, Y, and W residues.

8. PeptideMap PeptideSort Cleave polypeptide with proteases, reagents Predict HPLC properties of peptide fragments

9. Protein Analyses Secondary Structure Prediction Hydrophobicity/membrane insertion Antigenicity Surface Accessibility Baylor Search Launcher: http://searchlauncher.bcm.tmc.edu/seq-search/struc- predict.html ExPASy: http://www.expasy.ch/tools/

10. Secondary Structure Prediction Amino acid preferences Local aa interactions Non-local interactions Homology/Multiple alignments

11. Predict H, E, L (Helix, B-strand, Loop) ~75% aa accuracy Programs –APSSP –Jpred –PHDsec –SAM-T99 –PredictProtein Web sites –BCM Search Launcher –ExPASy Tools Secondary Structure Prediction

12. Membrane Association Hydropathy, Hydophobicity –  G transfer (water-vapor) –% sidechains buried (100% or 95%) Identify membrane-associated regions Identify membrane-spanning regions (>16 aa, ~80 Å helix)

13. Hydrophobicity Membrane Topology Original paper: Kyte-Doolittle –J. Mol. Biol. (1978) 157:105-132 Further refinements: Hopp-Woods –Proc Natl Acad Sci U S A (1981) 78:3824 Gunnar von Heijne GES-scale (Goldman, Engelman, Steitz) –Engelman et al, 1982

14. SOAP (Kyte-Doolittle) Plots

15. Membrane Topology Hydrophobic helices Dipoles aligned Charges? What is the overall structure? External and internal domains?

16. Localization Signal sequences direct proteins –Usually N or C terminal signal sequences Targeted to: membrane, secretion, nucleus, mitochondria, chloroplast, lysosome, peroxisome, periplasm Program criteria: N-term motifs, aa composition, protein domains specific to locations, homology to proteins with experimentally determined locations. Accuracy varies with type of prediction, 50-80% Programs: –WoLF PSORT (eukaryotic), PSORTb (bacterial), PSORT (plant) –SUBLOC –TargetP

17. Surface Accessibility Emini et al (1985) 55:836 Can also calculate (for real) on a PDB structure via WWW http://www.bork.embl- heidelberg.de/ASC/scr1-form.html

18. Antigenicity Jameson and Wolf (CABIOS 4:181) –Sums secondary structure indices, surface accessibility, backbone flexibility –Many good epitopes are linear, surface loops Used when “picking” antigenic peptides

19. Transmembrane segment prediction Hydrophobicity Membrane association/topology Programs –PHDhtm –TopPred –TMHMM