Directed Evolution Lecture Notes PDF

Protein engineering Directed evolution 1 WEEK 19 06/12/2022 Dr. Anastasios Stavrou Based on Dr. Aldo Gutierrez lecture Learning objectives • Describe strategies to modify enzymes by the use of natural selection or mutagenesis methods (“directed evolution”) • Describe and provide example of...

Protein engineering Directed evolution 1 WEEK 19 06/12/2022 Dr. Anastasios Stavrou Based on Dr. Aldo Gutierrez lecture Learning objectives • Describe strategies to modify enzymes by the use of natural selection or mutagenesis methods (“directed evolution”) • Describe and provide example of enzymes that are altered and their uses 16 March 2023 2 Creating enzymes with new properties K1. Explain the principles of current mainstream biochemical techniques and their applications K4. Explore how debate informs concerns about the quality and sustainability of life at local and international scales S 1 . Select appropriate techniques to study the purification and analysis of proteins and peptides . S 2 . Analyse and evaluate biological data to determine equilibrium binding constants 3 Directed evolution of proteins: what all the fuss is about? Duration: 3 min The National Academy of Engineering (USA) annually awards the Charles Stark Draper Prize, which is given for the advancement of engineering and the education of the public about engineering. It is regarded as the "Nobel Prize of Engineering“. 16 March 2023 4 Prof. Frances Arnold (Caltech, USA) Pioneer of proteins directed evolution 1 - Fundamentals: From the genetic code to site - directed mutagenesis. March 16, 2023 5 16 March 2023 6 The genetic code 16 March 2023 7 Site -Directed mutagenesis Lecture Dr Sergio Colombo Site -directed mutagenesis opened a new way to study the role of single amino acids in binding sites. Ligand: cyclic AMP Binding site: Kinase A (PKA) 16 March 2023 10 "for fundamental contributions to the establishment of site -directed mutagenesis (SDM)” The Nobel Prize in Chemistry 1993 Michael Smith SDM method Kary B. Mullis PCR reaction 2 - Structural cooperativity and the need for global mutagenesis March 16, 2023 11 16 March 2023 12 BIOL22081 : 2 lectures on allosteric regulation (2 nd term, WEEKS 30&31) Biomolecular Structure and Function 16 March 2023 13 Example of structural cooperativity: Phosphofructokinase (PFK) . Propagation from one sub -unit to other . WEEK 30 BIOL22081 16 March 2023 14 Example of structural cooperativity: Dihydrofolate reductase. Mapping the propagation network . WEEK 30 BIOL22081 16 March 2023 15 A small 100 amino acid -long protein. The problem: structural cooperativity and mutagenesis 20 100 = 1.3 x 10 130 different mutants G D A N V E L Q I K S R T H C F M Y P W Each of the 100 amino acids can be replaced by 20 different amino acids 16 March 2023 16 The problem: structural cooperativity and mutagenesis 1.3 x 10 130 7 x 10 20 number of stars in the Universe 16 March 2023 17 20 100 = 1.3 x 10 130 different single directed mutagenesis experiments 16 March 2023 18 Directed evolution method: Directed evolution method: 1) Select target gene 2) Create a DNA library of mutants 3) Insert gene library / vectors into bacteria, which produce enzyme variants 4) Screen colonies for the property of interest 5) Isolate improved gene(s) and repeat the process. Directed evolution method: 1) Select target gene 2) Create a DNA library of mutants 3) Insert gene library / vectors into bacteria, which produce enzyme variants 4) Screen colonies for the property of interest 5) Isolate improved gene(s) and repeat the process. 16 March 2023 21 2 - Creating a library of MILLIONS of mutants. A) Error -prone PCR - Taq DNA polymerase lacking proof -reading activity - unbalanced nucleosides concentrations (A,G,C,T) - aberrant false nucleosides (e.g.8 -oxo -guanine) 16 March 2023 22 2 - Creating a library of MILLIONS of mutants. B) DNA shuffling A family of homologues genes are randomly fragmented and re -ligated creating “chimeric genes” 16 March 2023 23 A 16,500 plates microchip Current technical limit: 1 million mutants/ per day 4 - Screening colonies for the property of interest - There must be an optical property related to the activity looked for. - Automated robotics are fundamental (microchip assay) 16 March 2023 24 -Microchip assay: monitoring 16,500 reactions in real time Time (s) Reaction signal from 2 microplates: low activity high activity 16 March 2023 25 Now... what are we looking for?? 16 March 2023 26 Existing enzymes are already very fast Orotidine 5' -phosphate decarboxylase (OMP). Purine biosynthesis. 16 March 2023 27 One aim: Making industrial enzymes more stable 16 March 2023 28 The big prize: Creating enzymes with new properties 29 The astonishing power of enzyme catalysis Transition states For new bonds to be made….. ……old ones must be broken !!!! The Eyring -Polanyi equation relates the amount of activation energy required to bring the substrate to the transition state conformation with the rate constant of the entire reaction . ?????? = ?????? ?????? ?????? ?????? ?????? −∆??????≠ ???????????? ?????? = ?????? ?????? ?????? ?????? ?????? −∆??????≠ ???????????? 16 March 2023 32 An outstanding early example: Nature, 8 May, 2008 This article was chosen as one of the 10 most important scientific papers in 2008. A PDF copy of this article is in NOW 16 March 2023 33 Creating a novel enzyme from scratch: This Kemp elimination reaction is not required in cells, so there is not any enzyme to catalyse it. First step : they calculated how the TRANSITION STATE would look like in this reaction: Second step : Based on the hypothetical transition state , they deduced what kind of residues would be needed to reduce its activation energy. Charge stabilization TRANSITION STATE 16 March 2023 35 Four step : A synthetic DNA gene was made coding for TIM -barrel and “transition state -stabilizing” residues. This gene doesn’t exist in nature!!! Then, THEY APPLIED DIRECTED -EVOLUTION METHODS..... Third step : they used the TIM -barrel structure as an scaffold . TIM -barrel is a highly occurring structure shape (motif) in proteins. It is highly flexible. TIM - barrel 16 March 2023 36 ..... a new enzyme is born !!!!! Multiple turnover kinetics Binding site as predicted !! First step : they calculated how the TRANSITION STATE would look like in this reaction: Second step : Based on the hypothetical transition state , they deduced what kind of residues would be needed to reduce its activation energy. Charge stabilization TRANSITION STATE 16 March 2023 38 Binding site as predicted !! Charge stabilization 16 March 2023 39 2018 Chemistry Nobel Prize 16 March 2023 40 PDF file in NOW 16 March 2023 41 “New enzymes by evolution” Excellent talk (39 minutes) Conclusions • Describe strategies to modify enzymes by the use of natural selection or mutagenesis methods (“directed evolution”) • Describe and provide example of enzymes that are altered and their uses • Importance of activation energy 16 March 2023 42 Creating enzymes with new properties • Questions March 16, 2023 43