* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Career of Tom Muir
Immunoprecipitation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Biochemistry wikipedia , lookup
Index of biochemistry articles wikipedia , lookup
Gene expression wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Magnesium transporter wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Alternative splicing wikipedia , lookup
List of types of proteins wikipedia , lookup
Protein design wikipedia , lookup
Expression vector wikipedia , lookup
Interactome wikipedia , lookup
Protein domain wikipedia , lookup
Protein structure prediction wikipedia , lookup
Self-assembling peptide wikipedia , lookup
Protein moonlighting wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Western blot wikipedia , lookup
Protein folding wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Protein adsorption wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Peptide synthesis wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
The Career of Tom W. Muir Scott Simonovich MacMillan Group Meeting February 24th 2010 Previous Methods for Peptide Synthesis O support O O NH2 peptide NHBoc HO R R R' R' H N NHBoc O Solid phase peptide synthesis (SPPS) desire polypeptide cleavage O R' H N iterations R NH2 O ■ Useful method with significant drawbacks ■ Incorporate unnatural or D-amino acids ■ Each step must be very efficient 35 residue polypeptide: 99% yield per step = 49% IY 95% yield per step = 3% IY ■ Maximum length of ~50 amino acids ■ Can be very time consuming ■ Many peptides not suitable for SPPS Chemical Ligation S O O polypeptide 1 H3N SR HN polypeptide 2 RSH Native Ligation (NL) ■ Coupling of unprotected AA's O ■ Compatible with side chains polypeptide 1 S O H2N HN polypeptide 2 ■ Nearly quanitative yield ■ General tool for semi-synthesis ■ Works at physiological pH O polypeptide 1 SH HN H N ■ Numerous improvements polypeptide 2 O Figure adapted from Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776. Chemical Ligation ■ Applications in protein engineering to study biological systems ■ Insertion of synthetic peptide into recombinant protein ■ Biosensor with properties dependent upon state of system domain 1 SO2NH domain 2 (dansylamide) fluorescent probe NMe2 ■ Abelson nonreceptor protein tyrosine kinase (Abl) ■ src Homolgy 2 domain (SH3) and src Homolgy domain 2 (SH2) Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100. Chemical Ligation oligopeptide PG HS RGEIKGR Me2N N H S O H N O O N H SR' O H N O ligation site fluorophore Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100. Chemical Ligation HS RGEIKGR Me2N H N N H S O O O N H HS SR' O Abl-SH2 H2N H N O O Dns Abl-SH3 Abl-SH2 6 M GdmCl, 140 mM NaCl, 200 mM phosphate buffer 1.5 vol% benzylmercaptan, 1.5 vol% thiophenol, 96 hours Dns Dns HS RGEIKGR N H Factor Xa Abl-SH2 O deprotection HS O Abl-SH2 H2N Abl-SH3 SR' O Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100. Chemical Ligation ■ Kd = 0.123 0.017 µM ■ Monodentate ligands led to insignificant fluorescence increases ■ Useful biosensor for future investigations ■ High affinity, bidentate ligands ■ in vitro screening of combinatorial peptide libraries ■ Characterize protein-protein interactions that regulate Abl function domain 1 domain 2 fluorescent probe Can native ligation regulate molecular processes to study protein function? Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100. Chemical Ligation ■ Chemical modification of proteins + external impulse ■ Semi-synthesis through native ligation with photolabile PG ■ Traditionally difficult to prepare photocaged systems UV light polypeptide protein that controls cell function ligation polypeptide photorelease photocaged protein cell ■ Control of protein function through subcellular localization with light Pellois, J-.; Muir, T. W. Angew. Chem. Int. Ed. 2005, 44, 5713. Chemical Ligation required for ligation required for UV photolytic cleavage HS H3N NO2 H2N H N O OMe O HN O NH2 HN HN SO2 O O O3S required for fluorescence spectroscopy Texas Red N O N ■ Synthesize fluorophore-bearing fragment and ligate to recombinant protein Pellois, J-.; Muir, T. W. Angew. Chem. Int. Ed. 2005, 44, 5713. Chemical Ligation ■ Photolysis of protein in a cell ■ Dosable manner with low-intensity UV light ■ Valuable if different protein concentrations trigger different responses ■ Photolysis of small molecule can change function of protein O Abl-SH3 SR How are protein-bearing thioesters constructed? Total synthesis Expression O general methods protein Expressed Protein Ligation SR Expressed Protein Ligation (EPL) Protein Splicing SH O SH O N-Extein Intein N H C-Extein N H O ■ No sequence requirements on exteins NH2 O NH O O N-Extein Intein S HS N H O NH2 Intein SH C-Extein O NH2 NH2 N-Extein N-Extein Intein NH2 N H O NH2 N H C-Extein O S O HS SH O C-Extein Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249. Expressed Protein Ligation (EPL) N-Extein SH O O Intein S N H O NH2 NH2 N-Extein S O Intein HS NH2 N H O NH2 O C-Extein C-Extein Expressed Protein Ligation (EPL) SH O Intein HS Me NH2 O N-Extein C-Extein N H SPh HS peptide H2N O PhSH N-Extein SH O O Intein S N H Me NH2 SH O C-Extein N-Extein N H peptide O ■ Access to large proteins N-Extein Intein NH2 Me ■ General method S O HS O N H C-Extein ■ Post-translational modifications ■ Structure and functions Expressed Protein Ligation (EPL) Protein Conformation ■ Trp intrinsic fluorophore - sensitive to local environment Trp HN Trp R R O HN Trp ligand HN O Trp HN Trp Trp NH NH Trp ■ Risks of destabilizing protein or altering function ■ Trp analogs well suited for studying structure (lack of techniques) Trp Expressed Protein Ligation (EPL) Protein Conformation ■ Trp intrinsic fluorophore - sensitive to local environment Trp HN Trp R R O HN Trp O ligand HN Trp HN Trp Trp NH NH Trp Trp N N ■ Risks of destabilizing protein or altering function ■ Trp analogs well suited for studying structure (lack of techniques) ■ Dynamic properties of SH3 domain SH2 SH3 c-Crk-I ■ Previously studied in isolation ■ Domain-specific biophysical information Expressed Protein Ligation (EPL) O intein SH2 SH2 RSH SR α-thioester coupling partner recombinant expression HS 7AW His-tag SH3 Xa 7AW O HS SH2 7AW SH3 SR H2N 7AW 7AW H2N protease recombinant expression (E. coli Trp auxotroph) 7AW SH3 Terminal cys coupling partner native Trp ligation Trp 7AW SH2 SH3 7AW (6 M GdCl) c-Crk-I Muir, T. W.; et al. J. Am. Chem. Soc. 2004, 126, 14404. Small Molecule Protein Splicing ■ Temporal control over protein function with small molecules ■ Turning protein on/off is difficult with standard genetic techniques Chemical Genetics ligand ligand Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044. Small Molecule Protein Splicing ■ Temporal control over protein function with small molecules ■ Turning protein on/off is difficult with standard genetic techniques Chemical Genetics peptide peptide peptide peptide peptide peptide ■ Dramatic changes in primary structure lead to changes in function Conditional Protein Splicing Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044. Small Molecule Protein Splicing O SH O N-Extein SH O N H Intein N H O NH Intein HS C-Extein O NH2 NH2 SH O ■ Protein splicing occurs spontaneously N-Extein C-Extein N H Conditional Protein Splicing = Protein Splicing + Molecule-Induced Heterodimerization HO FKBP12 FRB MeO Me Me (rapamycin) N O Me FKBP12 O HO O O O O OH Me O MeO OMe Me FRB Me Me Small Molecule Protein Splicing Conditional Protein Splicing = Protein Splicing + Molecule-Induced Heterodimerization FKBP12 FRB inteinN inteinC peptide peptide peptide peptide protein splicing ■ Low affinity intein fragments ■ Artificially split VMA intein ■ MBP and His peptide fragments ■ Fragemnts expressed in E. coli ■ No reaction without rapamycin peptide peptide Figures adapted from Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044. Small Molecule Protein Splicing FKBP12 FRB VMAN VMAC MBP His MBP ■ First example of protein splicing by small molecule ■ MBP and His are model protein ■ No structural or sequence restrictions to exteins Is this technique limited to the coupling of two peptide fragments? His Small Molecule Protein Splicing tandem splicing peptide peptide peptide peptide peptide Potential difficulties: ■ Intein specificity ■ Timing of splicing events ■ Occurs under physiological conditions ■ Starting materials only required in low concentrations peptide Small Molecule Protein Splicing tandem splicing peptide peptide peptide peptide peptide peptide ■ Essentially nothing is known about molecular recognition DnaEN DnaEC naturally split intein ■ Why do naturally split intein spontaneously splice? ■ Suggests intrinsic affinity difference between two intein types Small Molecule Protein Splicing FKBP12 FRB VMAN VMAC peptide peptide (rapamycin) P(CH2CH2CO2H)3 (TCEP) DnaEN DnaEC peptide peptide peptide c-Crk-II protein peptide TCEP peptide peptide peptide (rapamycin) Small Molecule Protein Splicing FKBP12 FRB VMAN VMAC peptide peptide DnaEN DnaEC (rapamycin) P(CH2CH2CO2H)3 (TCEP) peptide peptide peptide c-Crk-II protein peptide ■ Splicing can occur simultaneously or in a stepwise fashion ■ Additional tags and protecting groups for easy isolation purification ■ Proficient with purified fragments or crude cell lysates ■ Streamlined process this great generality Summary N-Extein O O polypeptide 1 S O S O Intein HS H2N HN polypeptide 2 NH2 N H O C-Extein NH2 Native Chemical Ligation FKBP12 FRB VMAN VMAC MBP Expressed Protein Ligation His Conditional and Tandem Protein Splicing MBA His