Peptides in chemistry and biology 1. Phage display 2. Solid phase peptide synthesis and applications of synthetic libraries 3. Native chemical ligation in protein synthesis Scott and Smith, Science v. 249 (1990) p. 386 R1 R2 R3 R4 or SS R4 R3 R1 R2 HS O O N H N O O H N N H QuickTime™ and a Animation decompressor are needed to see this picture. NH2 O SH O H N O N HN O O H2N QuickTime™ and a Animation decompressor are needed to see this picture. NH S S O H N O O N HN O O NH S S H2N O O H N O N HN O O H2N NH S S O Fuzeon QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Science v. 271 p. 1854 (1996) Helical Wheels - to determine amphilphilicity Native chemical ligation --Steve Kent Solid phase peptide synthesis Cl Cl Cl 100-500 µ Cl 500 nm 15 Å HO Cl O O OH HO O O O Cs2CO3 acid-labile linker = HO Linker Fmoc Solid Phase Peptide Synthesis H N O O Coupling Agent OH Fmoc H N O O N N N O N Fmoc O H2N O Linker S H N H2N O Base O Linker O S Fmoc N H H N O O Linker O S O N H HNFmoc O O 1. Chain elongation (Fmoc peptide synthesis) 2. Allyl ester deprotection O H X3 O H X5 X N N N N NH2 2 4 O O H X O H X O NH N H O HO 1 O H2N 1. Cyclization (Peptide coupling) 2. Cleavage (acid) X1 H O X2 N O NH O HN X3 NH O HN O N 4 X5 O H X Gramicidin S - a cyclic peptide antibiotic Split pool synthesis A Linker A Linker B Linker B C Linker C Pool Linker Linker Linker A B C D A B Linker C AD Linker BD Linker CD E Linker Linker Linker A B C Split A B Linker C AE Linker BE Linker CE F A B C Linker AF Linker BF Linker CF a c ti v it y Positional scanning Linker Linker Linker AXX BXX CXX Linker Linker Linker XDX XEX XFX Linker Linker Linker XXG XXH XXI Most active predicted sequence: BDI How to identify kinase substrates? O HO P O O O kinase Signal output Positional scanning library Cantley et al. JBC v. 272 (1997) p. 952 OH PKC isoforms O O P O O O OH2 O Fe N O OH2 O OH O N O e F O O O O P O O Wash, sequence bound peptides Results Approaches to the study of protein function 1. Biochemistry 2. Genetics 3. Pharmacology http://bio.research.ucsc.edu/people/kellogg/ Approaches to the study of protein function 1. Biochemistry - requires cell-free assay 2a. Forward genetics mutagenize Screen for interesting phenotype Trace phenoptype to mutation 2b. Reverse genetics a. Directed deletions, point mutations b. Gene silencing (siRNA) c. Knock-out mice 3a. Forward chemical genetics Mayer et al., Science (1999) v. 286 pp. 971 - 974 control + monastrol Target ID: the candidate approach http://mc11.mcri.ac.uk/wro ngtrousers.html Kinesin movies http://www.proweb.org/ kinesin/ Reverse Chemical Genetics Kevan Shokat Curr. Op. Chem. Biol v. 5 p. 360 (2001) Ellman, et al.