1 Directed Mutagenesis and Protein Engineering

2 Mutagenesis Mutagenesis -> change in DNA sequence -> Point mutations or large modifications Point mutations (directed mutagenesis): -Substitution: change of one nucleotide (i.e. A-> C) -Insertion: gaining one additional nucleotide -Deletion: loss of one nucleotide

3 Consequences of point mutations within a coding sequence (gene) for the protein Silent mutations: -> change in nucleotide sequence with no consequences for protein sequence -> Change of amino acid -> truncation of protein -> change of c-terminal part of protein

4 Mutagenesis Comparison of cellular and invitro mutagenesis

5 Applications of directed mutagenesis

6 General strategy for directed mutagenesis Requirements: - DNA of interest (gene or promoter) must be cloned - Expression system must be available -> for testing phenotypic change

7 Approaches for directed mutagenesis -> site-directed mutagenesis -> point mutations in particular known area result -> library of wild-type and mutated DNA (site-specific) not really a library -> just 2 species -> random mutagenesis -> point mutations in all areas within DNA of interest result -> library of wild-type and mutated DNA (random) a real library -> many variants -> screening !!! if methods efficient -> mostly mutated DNA

8 Protein Engineering -> Mutagenesis used for modifying proteins Replacements on protein level -> mutations on DNA level Assumption : Natural sequence can be modified to improve a certain function of protein This implies: Protein is NOT at an optimum for that function Sequence changes without disruption of the structure (otherwise it would not fold) New sequence is not TOO different from the native sequence (otherwise loss in function of protein) consequence -> introduce point mutations

9 Protein Engineering Obtain a protein with improved or new properties Proteins with Novel Properties Rational Protein DesignNature Random Mutagenesis

10 Rational Protein Design Site –directed mutagenesis !!! Requirements: -> Knowledge of sequence and preferable Structure (active site,….) -> Understanding of mechanism (knowledge about structure – function relationship) -> Identification of cofactors……..

11 Site-directed mutagenesis methods Old method -> used before oligonucleotide –directed mutagenesis Limitations: -> just C-> T mutations -> randomly mutated

12 Site-directed mutagenesis methods

13 Site-directed mutagenesis methods – Oligonucleotide - directed method

14 Site-directed mutagenesis methods – PCR based

15 Directed Evolution – Random mutagenesis -> based on the process of natural evolution - NO structural information required - NO understanding of the mechanism required General Procedure: Generation of genetic diversity Random mutagenesis Identification of successful variants Screening and seletion

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17 General Directed Evolution Procedure Random mutagenesis methods

18 Directed Evolution Library Even a large library -> (10 8 independent clones) will not exhaustively encode all possible single point mutations. Requirements would be: 20 N independend clones -> to have all possible variations in a library (+ silent mutations) N….. number of amino acids in the protein For a small protein: -> Hen egg-white Lysozyme (129 aa; 14.6 kDa) -> library with (7x ) independent clones Consequence -> not all modifications possible -> modifications just along an evolutionary path !!!!

19 Limitation of Directed Evolution 1.Evolutionary path must exist - > to be successful 2.Screening method must be available -> You get (exactly) what you ask for!!! -> need to be done in -> High throughput !!!

20 Successful experiments involve generally less than 6 steps (cycles)!!! Why? 1.Sequences with improved properties are rather close to the parental sequence -> along a evolutionary path 2. Capacity of our present methods to generate novel functional sequences is rather limited -> requires huge libraries Point Mutations !!! Typical Directed Evolution Experiment

21 Evolutionary Methods Non-recombinative methods: -> Oligonucleotide Directed Mutagenesis (saturation mutagenesis) -> Chemical Mutagenesis, Bacterial Mutator Strains -> Error-prone PCR Recombinative methods -> Mimic natures recombination strategy Used for: Elimination of neutral and deleterious mutations -> DNA shuffling -> Invivo Recombination (Yeast) -> Random priming recombination, Staggered extention precess (StEP) -> ITCHY

22 Evolutionary Methods Type of mutation – Fitness of mutants Type of mutations: Beneficial mutations (good) Neutral mutations Deleterious mutations (bad) Beneficial mutations are diluted with neutral and deleterious ones !!! Keep the number of mutations low per cycle -> improve fitness of mutants!!!

23 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)

24 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)

25 Random Mutagenesis (PCR based) Error –prone PCR -> PCR with low fidelity !!! Achieved by: - Increased Mg2+ concentration - Addition of Mn2+ - Not equal concentration of the four dNTPs - Use of dITP - Increasing amount of Taq polymerase (Polymerase with NO proof reading function)

26 Random Mutagenesis (PCR based) DNA Shuffling DNase I treatment (Fragmentation, bp, Mn 2+ ) Reassembly (PCR without primers, Extension and Recombination) PCR amplification

27 Random Mutagenesis (PCR based) Family Shuffling Genes coming from the same gene family -> highly homologous -> Family shuffling

28 Random Mutagenesis (PCR based)

29 Directed Evolution Difference between non-recombinative and recombinative methods Non-recombinative methods recombinative methods -> hybrids (chimeric proteins)

30 Protein Engineering What can be engineered in Proteins ? -> Folding (+Structure): 1. Thermodynamic Stability (Equilibrium between: Native Unfolded state) 2. Thermal and Environmental Stability (Temperature, pH, Solvent, Detergents, Salt …..)

31 Protein Engineering What can be engineered in Proteins ? -> Function: 1. Binding (Interaction of a protein with its surroundings) How many points are required to bind a molecule with high affinity? 2. Catalysis (a different form of binding – binding the transition state of a chemical reaction) Increased binding to the transition state increased catalytic rates !!! Requires: Knowledge of the Catalytic Mechanism !!! -> engineer Kcat and Km

32 Protein Engineering Factors which contribute to stability: 1. Hydrophobicity (hydrophobic core) 2. Electrostatic Interactions: -> Salt Bridges -> Hydrogen Bonds -> Dipole Interactions 3.Disulfide Bridges 4.Metal Binding (Metal chelating site) 5.Reduction of the unfolded state entropy with X Pro mutations

33 Protein Engineering Design of Thermal and Environmental stability: 1. Stabilization of -Helix Macrodipoles 2. Engineer Structural Motifes (like Helix N-Caps) 3. Introduction of salt bridges 4. Introduction of residues with higher intrinsic properties for their conformational state (e.g. Ala replacement within a -Helix) 5. Introduction of disulfide bridges 6. Reduction of the unfolded state entropy with X Pro mutations

34 Protein Engineering - Applications Engineering Stability of Enzymes – T4 lysozyme -> S-S bonds introduction

35 Protein Engineering - Applications Engineering Stability of Enzymes – triosephosphate isomerase from yeast -> replace Asn (deaminated at high temperature)

36 Protein Engineering - Applications Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from B. stearothermophilus -> replace Thr 51 (improve affinity for ATP) -> Design

37 Protein Engineering - Applications Engineering Ca-independency of subtilisin Saturation mutagenesis -> 7 out of 10 regions were found to give increase of stability Mutant: 10x more stable than native enzyme in absence of Ca 50% more stable than native in presence of Ca

38 DNA shuffling JCohen. News note: How DNA shuffling works. Sci 293:237 (2001) Maxygen, PCR without synthetic primers Using family of related genes, digest into fragments Heat and renature randomly Use as PCR primers

39 Altering multiple properties: rapid high- throughput screening ex., subtilisin Use 26 different subtilisin genes Shuffle DNA, construct library of 654 clones, and Tf B. subtilis Assay in microtiter plates: originals plus clones Activity at 23C; thermostability; solvent stability; pH dependence Of 654 clones, 77 versions performed as well as or better than parents at 23C Sequencing showed chimeras; one has 8 crossovers with 15 AAc substitutions

40 Laundry, detergent and mushrooms Peroxidase, ink cap mushroom; dye transfer inhibitor Wash conditions: bleach-containing detergents, pH 10.5, 50C, high peroxide concentration (inactivates peroxidase) Random mutagenesis or error-prone PCR, followed by DNA shuffling One construct had 114x increase in thermal stability, 2.8x increase in oxidative stability

41 ex., Coprinus cinereus heme peroxidase (ink cap mushroom); 343 AAc, heme prosthetic group Multiple rounds of directed evolution to generate mutant for dye transfer inhibitor in laundry detergent Native form or WT is rapidly inactivated under laundry conditions at pH 10.5, 50C and high peroxide concentrations (5-10mM) Combined mutants from site-directed and random mutagenesis led to mutant with 110x thermal stability, 2.8x oxidative stability Additional in vivo shuffling of pt mutations -> 174x thermal stability and 100x oxidative stability Cherry…Pedersen. 99. Nat Biotech Directed evolution of a fungal peroxidase Mushroom peroxidase

42 Molecular analysis of hybrid peroxidase

43 Decreasing protein sensitivity Streptococcus streptokinase, 47 kDa protein that dissolves blood clots Complexes with plasminogen to convert to plasmin, which degrades fibrin in clots Plasmin also degrades streptokinase [feedback loop] In practice, need to administer streptokinase as a min infusion [heart attacks] A long-lived streptokinase may be administered as a single injection www-s.med.uiuc.edu; JMorrissey: Med Biochem 10/30/06

44 Decreasing protein sensitivity Streptococcus streptokinase, plasmin sensitivity domain Attacks at Lys59 and Lys382, near each end of protein Resultant 328 AAc peptide has ~16% activity Mutate Lys to Gln Gln has similar size/shape to Lys also no charge Single mutations similar to double to native in binding and activating plasminogen; In plasmin presence, half-lives increased with double as 21x more resistant to cleavage TBD…(2003) longer life wanted

45 Protein Engineering - Applications Site-directed mutagenesis -> used to alter a single property Problem : changing one property -> disrupts another characteristics Directed Evolution (Molecular breeding) -> alteration of multiple properties

46 Protein Engineering – Applications Directed Evolution

47 Protein Engineering – Applications Directed Evolution

48 Protein Engineering – Applications Directed Evolution

49 Protein Engineering – Applications Directed Evolution

50 Protein Engineering – Directed Evolution

51 Protein Engineering - Applications

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