Protein engineering

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Credit Seminar : BIO Protein Engineering in Crop Improvement Presented By: Bhupendra Singh Seminar Leader: Dr Archana Singh Chairperson: Dr Sarvajeet Kaur

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INTRODUCTION Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Definition The design and construction of new proteins or enzymes with novel or desired functions by modifying amino acid sequences by using recombinant deoxyribonucleic acid technology. Protein engineering is the process of developing useful or valuable proteins. Protein engineering is the design of new enzymes or proteins with new or desirable functions. It is based on the use of recombinant DNA technology to change amino acid sequences

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INTRODUCTION The first papers on protein engineering date back to early 1980ies: in a review by Ulmer (1983), In 1992 a review by Gupta, protein engineering was mentioned as a highly promising technique within the frame of biocatalyst engineering to improve enzyme stability and efficiency in low water systems. Today, owing to the development in recombinant DNA technology and high-throughput screening techniques, protein engineering methods and applications are becoming increasingly important and widespread. In this saminar , a chronological review of protein engineering methods and applications in crop improvement is provided. Ulmer, KM. (1983). Protein engineering. Science, Vol. 219, No. 4585, (February 1983), pp.666-671, ISSN: 0036-8075 Gupta, MN. (1992). Enzyme function in organic-solvents. European Journal of Biochemistry, Vol. 203, No. 1-2, (January 1992), pp.25-32, ISSN: 0014-2956

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Objective of Protein Engineering The objectives of protein engineering is as follows – (a) to create a superior enzyme to catalyze the production of high value specific chemicals. (b) to produce enzyme in large quantities. (c) to produce biological compounds(include synthetic peptide, storage protein, and synthetic drugs) superior to natural one.

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In view of above, the enzyme should be engineered to meet the altered needs. Therefore efforts have been made to alter the properties of enzymes. These are some character that one might have to change in a predictable manner in protein engineering or enzyme engineering to get the desired function :- Objective of Protein Engineering Kinetic properties of enzyme-turnover and Michaelis constant, Km. Thermo stability and the optimum temperature for the enzyme. Stability and activity of enzyme in nonaqueous solvents. Substrate and reaction specificity. Cofactor requirements Optimum PH. Molecular weight and subunit structure. Therefore for a particular class of enzymes, variation in nature may occur for each of the above properties, so that one may like to combine all the optimum properties to the most efficient form of the enzyme.

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Basic Assumption for Protein Engineering While doing protein engineering should recognize the following properties of enzymes, many amino acid substitution, deletions or additions lead to no changes in enzyme activity so that they are silent mutator. Protein have limited number of basic structures and only minor changes are superimposed on them leading to variation Similar patterns of chain folding and domain structure can arise from different amino acid sequences with little or no homology.

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Protein Engineering Methods There are two general wet lab strategies for protein engineering, rational design and directed evolution . Directed Evolution 3D structure Replace amino acids Site-directed Mutagenesis/transformation And expression Characterization Mutant proteins Screen and select best candidate Characterization ePCR Shuffling Mutant Genes Transform and express Single Gene Gene family Rational Engineering

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Rational Redesign Strategy Analysis of Protein structure Planning of mutants (Site directed mutagenesis) Vector contain mutated genes Transformation in E. coli Protein expression and purification Mutant enzyme analysis Negative mutants Improved mutant analysis

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Rational Redesign Strategy Geometry Based Design Stereochemistry based Design Peptide Torsion Angles Distortions of alpha-helices Beta-Sheet Geometry Ramachandran Plot Reverse Turns Beta-Hairpin turns Side Chain Conformation. Side Chains or R-groups Axis of rotation Angle of rotation Direction cosines of the axes of rotation Minimum contact distance assumed Preferred orientations of polypeptide

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What is Directed Evolution? An engineering strategy used to improve protein functionality through repeated rounds of mutation and selection First used in the ‘70s Around .01-1% of all random mutations estimated to be beneficial Based on natural evolution processes, but in a much quicker timescale Direct Evolution

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Direct Evolution

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Direct Evolution Random Mutagenesis Traditional method Point mutation based – error prone PCR Frequency of beneficial mutations very low Multiple mutations virtually impossible to come out positive Error prone RCA Error prone PCR Mutator strain Error prone RCA Error prone PCR Transformation of mutator strain Digestion with restriction enzymes Separation of fragments Extraction of plasmid DNA Ligation Transformation Transformation Transformation

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DNA Shuffling Recombination used to create chimeric sequences containing multiple beneficial mutations “Family shuffling” of homologous genes “Synthetic shuffling” – oligonucleotides combined to create full-length genes Direct Evolution Whole-genome shuffling – accelerated phenotypic improvements Drawback – high homology required DNA Shuffling Hybrid Genes Species 1 Species 2 Species 3 Ancestral gene Natural Evolution

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RACHITT Ra ndom Chi meragenisis on T ransient T emplates Small DNA fragments hybridized on a scaffold to create a chimeric DNA fragment Incorporates low-homology segments Direct Evolution Library of randomly mutated genes or pool of homologous gene Random fragmentation by digest with DNaseI Wild type single strand Hybridization Remooval of flaps and fill in reaction by pfu polymerase Removal of template strand by uracil-DNA-glycosylase Generation of dsDNA by PCR

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Even More Methods Assembly of Designed Oligonucleotides (ADO) Mutagenic and Unidirectional Reassembly (MURA) Exon Shuffling Y-Ligation-Based Block Shuffling Nonhomologous Recombination – ITCHY, SCRATCHY, SHIPREC, NRR Combining rational design with directed evolution Direct Evolution

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Computational Protein Design A Blueprint of CPD Approaches Property/Activity Biological databases Dataset Selection Structure Based Sequence Based Force fields Knowledge based potentials Entropic factors (RS1) Identification of hot spot modular regions (RS3)(RS4) Topological Properties (Modularity) (RS2) Homology based models Signature based models (RS5) Knowledge based potentials

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Computational Protein Descriptors Molecular Signature Descriptors A 2D representation of the molecular graphs as an undirected colored graphs G(V, E, C), with V : atoms, E : bonds, C : atom type The signature descriptor of height h of atom x in the molecular graph G, or h σ (x), is a canonical representation of the subgraph of G containing all atoms that are at distance h from x Atomic signature : The signature is a systematic codification of the molecular graph [Faulon et al., 2004] L-31 σ (methylcyclopropane) = 1 [C]([H][C]([H][H][C,0])[C,0]([H][H])[C]([H][H][H])) 2 [C]([H][H][C]([H][C,0][C]([H][H][H]))[C,0]([H][H])) 1 [C]([H][H][H][C]([H][C]([H][H][C,0])[C,0]([H][H]))) 1 [H]([C]([C]([H][H][C,0])[C,0]([H][H])[C]([H][H][H]))) 4 [H]([C]([H][C]([H][C,0][C]([H][H][H]))[C,0]([H][H]))) 3 [H]([C]([H][H][C]([H][C]([H][H][C,0])[C,0]([H][H]))))

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Computational Protein Descriptors Molecular Signature of Reactions and Proteins Signature of a reaction. The signature of reaction R that transforms n substrates into m products is given by the difference between the signature of the products and the signature of the substrates: Signature of protein sequences. The protein P is represented by the linear chain given by its collapsed graph at residue level, a reduced molecular graph representation G(V; E;C) known as string signature where V : residues a ⋲A, E : contiguous in sequence, C : amino acid type

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Computational Protein Descriptors Protein Contact Maps The protein contact map is a graph representation of the 3D interactions at residue level G(V; E;C) where V : residues, E : contacts, C : amino acid type Two residues are considered to interact when atoms between both residues are at a distance lower than a predetermined threshold (tipically 4:5 5 Å) Contact maps can account for long-range interactions and conformational states

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Search Algorithms in CPD In SILICO Determinants Sequence descriptors Structure descriptors Energy function SEARCH ALGORITHMS Deterministic Dead-end elimination Magic-bullet metric Conformational splitting Background optimization Stochastic MCSA Genetic algorithms Strategies SCHEMA OPTCOM IPRO

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De Novo-Designed Proteins In de novo designs, some assumptions are needed in order to make the search space tractable Usually we start from some basic motifs or domains as scaffolds for the design Examples: motif resembling a zinc finger 3 and 4 helix bundles Helical coiled-coils Helix bundle motifs can be parametrized using a few global variables that describe the global structure Applications: New metal-binding sites Nonbiological cofactors for novel biomaterials and electromechanical devices Novel enzymatic activities

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Achievements of Protein Engineering

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Rational Redesign for Substrate Specificity Proc. Natl. Acad. Sci. USA Vol. 94, pp. 4872–4877, May 1997 Biochemistry Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position (unsaturated fatty acidy / diiron / nonheme iron / rational design / protein engineering) EDGAR B. CAHOON*, YLVA LINDQVIST†, GUNTER SCHNEIDER†, AND JOHN SHANKLIN*‡ Δ 9 -18:0-ACP desaturase 30 amino acid domain encompassing residues 178-207

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DPGADNNPYLAYIYTSYQERATAISHGSLG __RT_______GF____F_____F____NTA Δ 9 Δ 6 Δ 6 Δ 6 Δ 9 1 178 207 355 Chimera 1 DPGADNNPYLAYIYTSYQERATAIS __RT_______GF____F_____F_ Δ 9 Δ 6 Δ 6 Δ 6 Δ 9 1 178 202 355 Chimera 2 Enzyme* Specific activity, nmol /min per mg protein Ratio of total specific activity 16:0-ACP:18:0-ACP 16:0-ACP 18:0-ACP Δ 6 Δ 9 Δ 6 Δ 9 Δ 6 -16:0-ACP desaturase (Wild type) 100±3 ND 11±1 5.5±0.2 6:1 Chimera 1 (Δ 6/ Δ 9 178-207 / Δ 6) 13 4.0 15 17 1:2 Chimera 2 (Δ 6/ Δ 9 178-202 / Δ 6) 227±25 ND 253±25 5.3±0.5 1:1

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These is a first step toward the design of acyl-ACP desaturase for the production of novel monosunsaturated fatty acids in transgenic oilseed Crops

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Rational Redesign for Nutritional Enhancement Protein Science (2000), 9:1642–1650. Cambridge University Press. Printed in the USA. Copyright © 2000 The Protein Society A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein KEITH R. ROESLER and A. GURURAJ RAO Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, Iowa 50131-1004 The aim of this study was to design stable proteins enriched in essential amino acids such as Lys, Met, Trp , and Thr Retention of thermodynamic and proteolytic stability was a key prerequisite because unstable proteins were not expected to accumulate to useful levels in plants. The free amino acids resulting from degradation of unstable proteins could be catabolized in plants or could cause feedback inhibition of amino acid biosynthesis either of which would limit nutritional enhancement.

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Barley chymotrypsin inhibitor-2 (CI-2) ? This protein is one of the relatively few plant proteins with a known three-dimensional (3D) Structure Sequences are available for numerous CI-2 homologs, giving clues about conserved residues that would be unlikely to tolerate substitutions CI-2 is a small, monomeric, single domain protein that has been the subject of many biochemical studies

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Amino acid sequences of WT and engineered CI-2 compared with 19 CI-2 homologs from diverse plant species. Red residues were identical to CI-2, and green residues were homologous. Blue residues were substitutions made in the CI-2 derivatives BHL5, BHL6, and BHL8.

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Residue no. WT amino acid Substitution Structural element 19 Asn Lys N-terminus 20 Leu Met N-terminal 22 Thr Cys β-strand 23 Glu Thr β-strand 31 Ser Thr N-capping 34 Glu Lys α-helix 38 Val Met α-helix 40 Leu Met α-helix 41 Gln Lys α-helix 47 Gln Lys β-strand 49 Ile Met β-strand Residue no. WT amino acid Substitution Structural element 56 Ile Lys Reactive-site loop 59 Met Gly Reactive-site loop 61 Tyr Trp Reactive-site loop 62 Arg Lys Reactive-site loop 63 Ile Met Reactive-site loop 69 Phe Trp β-strand 73 Leu Lys Turn 75 Asn Lys β-strand 78 Glu Lys Bend 79 Val Thr β-strand 81 Arg Lys β-strand 82 Val Cys β-strand

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3D structure of BHL8 showing amino acid replacements and sites. Protein Δ G H 2 O (kcal mol-1) ΔΔ G (kcal mol -1 ) M (kcal mol -1 M -1 ) [ GdmCl ] 50 % (M) WT CI-2 7.52±0.52 Nil 1.95±0.15 3.86±0.02 BHL5 2.20±0.23 4.59 1.67±0.11 1.32±0.05 BHL6 3.09±0.08 3.84 1.74±0.06 1.78±0.01 BHL8 6.96±0.72 0.49 1.93±0.21 3.61±0.02 Equilibrium unfolding parameters

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Rational Redesign for Novel molecules for Health and Nutrition Chemistry & Biology Brief Communication Structure-Based Engineering of Strictosidine Synthase: Auxiliary for Alkaloid Libraries Elke A. Loris,1 Santosh Panjikar,2 Martin Ruppert,1 Leif Barleben,1 Matthias Unger,3 Helmut Schu¨ bel,4 and Joachim Sto¨ ckigt1,5,* The Pictet-Spengler condensation is a well known reaction required for the chemical synthesis of alkaloids and represents the first dedicated step of indole alkaloid biosynthesis This reaction gives three structurally diverse and therapeutically potent alkaloid families 1 st :- 6000 isoquinoline structure (Morphine and codeine) 2 nd : 2000 structurally complex and important members of monoterpenoid indol alkaloid (vinblastine and vincristine) 3 rd :- small family of monoterpenoid isoquinolines (emetic drug emetine)

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Rational Redesign for Novel molecules for Health and Nutrition Strictosidine synthase (STR1) catalyzes the Pictet-Spengler condensation of tryptamine and secologanin and represents one of the most highly characterized enzymes of alkaloid biosynthesis, 5-methoxy- or 5-methyltryptamine is not accepted by native STR1. Positions 5 and 6 of tryptamine seem to be, crucial for pharmacological activity, and a number of valuable drugs, such as vinblastine, vincristine, reserpine, or quinine, harbor a methoxy group at these positions.

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Rational Redesign for Novel molecules for Health and Nutrition Substrates Enzymes K m (mM) K cat (S -1 ) K cat /K m (mM -1 S -1 ) Tryptamine Wild Type 0.072 10.65 147.92 Val208Ala 0.219 54.09 246.99 5-Methyl-tryptamine Wild Type nd - - Val208Ala 0.281 6.56 23.35 5-Methoxy-tryptamine Wild Type nd - - Val208Ala 3.592 79.66 22.18 6-Methyl-tryptamine Wild Type 0.393 2.32 5.90 Val208Ala 0.762 10.95 14.37 6-Methoxy-tryptamine Wild Type 0.962 5.32 5.53 Val208Ala 0.307 16.66 54.27 Kinetic Data for Conversion of Tryptamine and Its 5- and 6-Substituted Derivatives for the Wild-Type and Mutant STR1

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Protein engineering in Designed Mutation Breeding Gene Targeting Applied to Designed-mutation Breeding of High-Tryptophan Rice Hiroaki Saika and Seiichi Toki Dec 2011 Tryptophan (Trp) is one of the limiting essential amino acids in cereals such as rice, which is a major source of human food and livestock feed worldwide Anthranilate synthase (AS) is vital to the biosynthesis of Trp, and the α subunit of AS is susceptible to feedback inhibition by Trp or its analogue 5-methyl-Trp (5MT) Kanno, T. et al. Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway. Plant Physiol. 138, 2260-2268 (2005).

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Protein engineering in Designed Mutation Breeding There are no reports of natural rice varieties harboring mutated OASA2. OASA2 is one of the two AS α subunit isoforms in rice, mutations in which confer Trp insensitivity and enhance the catalytic activity of AS Exprementally they shows that free Trp accumulated to high levels in transformed rice calli over-expressing modified OASA2 On the basis of result of Kanno et al they selected two types of mutations, S126F/L530D and Y367A, showing different enzymatic kinetics

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Protein engineering in Designed Mutation Breeding Information obtained from in silico and in vitro data Target Gene Desirable Mutation Precision Mutagenesis via GT Designed mutation breeding development of genetic resources

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Protein engineering in Designed Mutation Breeding Trp contents in seedlings and mature seeds of both homozygous and heterozygous plants #3–4 and #5–11 were higher than in the original cultivar In both lines #3–4 and #5–11, because of the complete elimination of Trp-sensitive OASA2, the Trp content in homozygous seedlings was higher than that in heterozygous seedlings. The mature seeds of homozygous #3–4 plants accumulated 230 fold higher Trp than in non-transformants. A daily consumption of 280 g of these rice grains is estimated to meet the daily requirement for Trp in an adult human weighing 60 kg. The contents of all amino acids, including Trp, in mature seeds of homozygous #3–4 plants were higher than those in non-transformants. However, the ratio of other amino acids, including phenylalanine and tyrosine, synthesized in the branched pathway of Trp biosynthesis, to total amino acids was unchanged, suggesting that Trp can be accumulated specifically using this strategy.

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Protein engineering for disease resistance Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. N Asparagine D Aspartic acid G Glycine H Histidine I Isoleucine K Lysine S Serine

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Protein engineering for disease resistance

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Protein engineering for disease resistance A C T G/A

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High-efficiency TALEN-based gene editing produces disease-resistant rice T. Li., B. Liu., M. Spalding, D. Weeks, and B. Yang. 2012 . Nature Biotechnology 30:390-392. Protein engineering for disease resistance They used to TALEN technology to edit a genome of rice for bacteria blight resistance Condition for disease development Os11N3 gene Nodulin 3 Xanthomonas Oryzae Vr Xa7 Interaction Disease Development

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Os11N3 gene Disrupted Protein Xanthomonas Oryzae Vr Xa7 No Interaction Resistance Protein engineering for disease resistance Os11N3 gene TALNS TALNS Disrupted Gene

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The modification of natural enzymes and proteins by protein engineering is an increasingly important scientific field. Protein engineering applications cover a broad range of, crop improvement, biocatalysis for food and industry, as well as medical, environmental and nanobiotechnological applications. The synergistic power of rational design, computation, and directed evolution on the one hand, and parallel advances in plant breeding/plant sciences and the omics technologies on the other, offer unprecedented opportunities for genetic engineering of novel traits into the next generation of crop plants Conclusion

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Thank You