RATIONAL DESIGN. Protein Engineering Approaches. ! Site-directed mutagenesis

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1 Protein Engineering Approaches! Rational design! Site-directed mutagenesis! Directed evolution! Error-prone PCR (random mutations)! DA shuffling (followed by screening)! Selection procedures! Phage display! De novo design! Solid phase synthesis of peptides! Biological libraries 1 RATIOAL DESIG 2

2 Rational design and directed evolution STRUCTURE-BASED DESIG Analysis of 3D structures/models DIRECTED EVOLUTIO SITE-DIRECTED MUTAGEESIS RADOM MUTAGEESIS Error prone PCR / Homologous gene family DA SEQUECIG RECOMBIATIO DA shuffling EXPRESSIO FUCTIOAL SCREEIG OF LIBRARY Selection (in vivo) / Screening (in vitro) FUCTIOAL CHARACTERISATIO DA SEQUECIG STRUCTURAL DETERMIATIO FUCTIOAL CHARACTERISATIO RATIOAL DESIG STRUCTURAL DETERMIATIO 3 RATIOAL DESIG! Pre-requisite: understanding the structure-function relationship! Structural information is fundamental.! Process:! identification of structurally important residues! definition of the interactions! implications to their functions! sdm! functional characterisation! When no structure is available:! sequence homology! biochemical evidences! random scanning! Output:! identification of functionally important residues! identification of protein-ligand interactions 4

3 Human P450 5 Identification of functionally important residues! Site-directed mutagenesis.! Prerequisites:! the gene is cloned! it can be expressed in good yields (mgs)! the host cells do not produce a wt protein! Types of possible mutations:! insertion of one or more AA! deletion of one or more AA! replacement of one residue! Two methods:! 1. oligonucleotide-directed mutagenesis! 2. mutagenesis by PCR! 1. Oligonucleotide-directed mutagenesis:! oligo.: primer of synthetic DA encoding the mutation! Use of dut - ung - strains of E.coli to produce uracil-containing ssda template. Use of uracil -glycosylase to remove wt strand in ung + strains.! Use of incorporation of thio-nucleotides in mutant strand, to resist restriction endonucleases. 6

4 Multiple cloning site Plasmid vector Cloning Gene insert Denature and anneal oligonucleotide Mutagenic oligonucleotide Extend with DA polymerase Ligate with T4 DA ligase Select mutant Tranform in expression host 7! 2. Mutagenesis by PCR! Taq (Thermus aquaticus) polymerase! ecessary:! deoxynucleotides (dtps)! DA polymerase! primers! thermocycles:! heating/cooling cycles! Exponential growth:! Cycle n. Target mol.! 1 0! 5 8! ! ! ,144! 25 8,388,608! ,435,456! Steps: 8

5 A CODIG 5' R 1 3' PRIMER 2 PRIMER 1 O-CODIG 3' 5' R 2 B PCR1 CODIG 5' PRIMER 2 R 1 PRIMER 1 3' O-CODIG 3' R 3 5' PCR2 CODIG 5' PRIMER 2 R 3 MEGA-PRIMER R 1 3' O-CODIG 3' 5' R

6 Quik-change method Plasmid vector with gene and site for mutation Denature the plasmid and anneal with mutagenic primers Extend and incorporate themutagenic primers using Pfu Turbo Digest the methylated wild type DA with Dpn I Mutagenic primers»taq (Thermus aquaticus) polymerase»ecessary: deoxynucleotides (dtps) DA polymerase primers thermocycles: heating/cooling cycles»exponential growth: Cycle n. Target mol , ,388, ,435,456 Mutated plasmidcontaining nicked circular strands Mutated plasmid repaired by XL1-Blue Dpn I: 5 -Gm 6 ATC-3 11 Interesting new functions or artefacts?! Interesting data on function:! functional residue?! non-specific disruption?! Complementary methods:! solubility and stable expression! X-ray crystallography! multidimensional MR! thermodynamic analysis! circular dichroism spec.! fluorescence spec.! UV-vis spec.! EPR spec.! FT/IR spec.! monoclonal antibodies 12

7 Rational structural-functional approach! 3D structure must be known! Good for studies of protein complexes with, for example:! substrates (tyrosyl-tra synthetase)! ligands (maltose binding protein)! inhibitors (drug design)! Interactions between AA and substrate/ligand/inhibitor:! H-bonds! ion-pairs! hydrophobic interactions 13 Tyrosyl-tRA synthetase! Two different domains:!/" +!! Crucial step in protein synthesis: connect each aa to its RA t! 1st the aa is activated by ATP to give an enzyme-bound aa-adenilate! 2nd the complex is attacked by the RA t to give the amino-acyl- t RA! The 3D structure of the enzyme specific for tyrosine was solved by David Blow in London at 2.7 Å:! 320 aa, last 100 aa disordered! The!/" (red and green) binds ATP and tyrosine; the! (blue) function unknown 14

8 15 Analysis of tyrosyl-tra synthetase Wells and Fersht, (1985) ature, 316:

9 Analysis of the ligand-binding cleft of the MBP 17 Alanine scanning mutagenesis 18

10 The MBP as biosensor MBP-OPE S337C mutant MBP-CLOSED S337C mutant FLUOROPHORE MALTOSE 19 Fluorescence emission IABD E ester -((2-(iodoacetoxy)ethyl)--methyl)- amino-7-nitrobenz-2-oxa-1,3-diazole H3C CH2CH2 - O - C - CH2 - I O O Fluorescence Intensity µm maltose 0 µm maltose % Increase Fluor. Int Fitted curve # ex = 480 nm K d = 62 ± 0.2 µm O Emission wavelength / nm Maltose conc. / µm Acrylodan 6-acryloyl-2-dimethylaminonaphthalene µM maltose 50 (a) Fitted curve Me 2 O C C H CH2 Fluorescence Intensity µm maltose % Increase Fluor. Int # ex = 363 nm K d = 0.80 ± 0.01 µm Emission wavelength / nm Maltose conc. / µm (b) 20

11 Immobilisation on glass! Fluorescence microscopy: [A] Glass [B] Glass-S337C-BD [C] Glass-S337C-BD saturated maltose 21 Time-resolved fluorescence 22

12 Molecular basis:! Interconversion between 2 conformers/states: Time-resolved fluorescence of S337C-BD o maltose Saturating maltose Circular dichroism Lifetimes / ns Lifetimes / ns! TICT: Twisted Intramolecular Charge Transfer. To have efficient relaxation, donor/acceptor must be co-planar. O R CH 2 CH 3 O O 23 Another example: P ASA (A2) C62 C62-S C156 C156-S C400 C400-S CYS residues 24

13 Reading list:! Gilardi, G., Mei, G., Rosato,., Finazzi-Agro, A. and Cass, A.E.G. Spectroscopic properties of an engineered maltose binding protein, Protein Engineering 10, (1997), ! Gilardi, G., Fantuzzi, A. "Manipulating redox systems: applications to nanotechnology ", Trends in Biotechnology 19 (11) (2001), ! Soong, R.K. et al. Powering and inorganic nanodevice with a biomolecular motor, Science 290 (2000),