Protein engineering

determining the structure of protein then how we can engineer it

1. Protein Engineering To Elucinate & Improve Stability Presented by: Sabahat Ali (BS BIOCHEMISTRY) PMAS AAUR

2. Contents Introduction How we get protein variety? Basic assumptions Homology & protein engineering Objectives of protein engineering Methods of protein engineering Factors affecting stability Applications

3. INTRODUCTION What is protein engineering? The modification of protein structure with recombinant DNA tech or chemical treatment to get desirable function for better use in medicine, industry & agriculture.

4. Protein engineering is merging of several disciplines like : Molecular biology Protein chemistry Enzymology

5. How we get protein variety? Variety in protein molecules because of Point mutation Exon shuffling Recombination Natural selection

6. Basic Assumptions: In protein engineering, we should recognize following properties of many Amino acids substitution, addition or deletion lead to no changes in enzyme activity so they are silent mutator. Protein have limited no of basic structure and limited no of change are subjected on them leading to variation.

7. Continued... Similar pattern of chain folding & domain structure can arise from different Amino acids sequences with little or no homology.

8. Homology & protein Engineering Homologous protein - similar structure & function -conserved structural core -variable loop regions Protein engineering & knowledge of protein structure Primary , secondary & tertiary structure.

9. Continued... Components of secondary structure are Alpha helix (helical structure 3.5 amino acids per turn) Beta sheets(compose of beta sgtrands)

10. Continued .. Knowledge of secondary structure is necessary for prediction of tertiary structure. Alpha , beta, alpha/beta domains ,their correct folding especially which A.acids residues are involved in a-helix & which are in b- strands.

11. Objectives of Protein Engineering To create a superior enzyme to catalyze the production of high value specific chemicals. produce Enzyme in large quantity produce biological comp(synthetic peptides & storage protein) that are superior to natural one .

12. Continued... Overall Aim of Protein Engineering application is to get functionally more useful ✓Enzymes ✓Antibodies ✓Hormones ✓Receptors

13. Study of 3D-structure of protein ♦Obtained by information from →X-ray crystallography →NMR Spectroscopy

14. METHODS OF PROTEIN ENGINEERING ANZA ABBAS

15. A variety of methods are used in protein engineering such as Mutagenesis Recombinant DNA technology

16. Mutagenesis Mutagenesis → change in DNA sequence This may include point mutations or large modifications substitution: change of one nucleotide (i.e A→C) insertion: gaining of one additional nucleotide deletion: loss of one nucleotide

17. Gene modification: The two process of gene modification are In-vitro mutagenesis using synthetic oligonucleotides Synthesis of complete modified gene de-novo

18. Increase Stability & biological activity through various ways Addition of Sulfide bonds Changes of Asparagine to other Amino acids Reducing the free sulfhydral groups Single Amino acid changes

19. Addition of Sulfide Bonds › Significantly Increase in the stability of Enzymes. ›However additional disulfide bonds should not interfere with normal enzyme function. ›New protein with added disulfide bonds does not readily unfold at high temp. Examples: Xylanase

20. Change of Asparagine to other Amino Acids If asparagine & glutamine present in protein when heated, NH3 is released. Amino acids are converted to aspartic acid & glutamic acid. Protein may Refold & loss of activity. Example: Triosephosphate Isomerase

21. Reducing the free sulfhydral groups The protein or enzyme stability & activity can be increased by reducing the no of sulfhydral groups. Convert Cys to another amino acid(serine) Reduce dimerization Maintain activity of Enzyme Example: Human B- interferons

22. Single Amino acid changes The recombinant proteins can be improved in their stability and biology activity. This can be frequently achieved by single Amino acids changes. Examples: Insulin

23. b. Synthesis of complete modified Gene denovo Complete Gene in some cases have been chemically synthesized in the form of several Oligomers e.g Gene for insulin that are ligated in correct order to produce a comply Gene.

24. Chemically modification of Enzyme The protein synthesized after transcription undergo post translational modification. This leads to stability, structure integrity, altered solubility of individual proteins.

25. Continued.. Example: An enzyme L-asparginase has antitumour properties but is toxic with a life time of less then 18 hrs thus reducing its utility .

26. Continued.. L-asparginase can be modified by polyethene glycol derivatives which differ from the native Enzyme in the following way it retains only 52% of the catalytic activity of native it become resistent to proteolytic degradation it doesn't cause allergy.

27. Continued.. Example : Insulin - contain A& B chain linked by C-peptide of 35 Amino acids. It was shown that a sequence of 6 Amino acids for C-peptide was adequate for the linking function.

28. Two strategies of protein Engineering : Directed mutagenesis Rational protein design

29. Directed mutagenesis

30. Site Directed mutagenesis Point mutation in particular gene Results in wild type and mutated copy of that gene

31. 2. Random mutagenesis point mutation in all areas within DNA of interest Result library of wild type and mutated DNA( random) a real library many variants →screening

32. Rational protein design knowledge of sequence and preferable structure (active site) Understanding of mechanism ( Knowledge about structure -function relationship) Identification of cofactors ........

33. What can be engineered in proteins? Structure (Folding) 1- Thermodynamic stability (Equilibrium between Native and Unfolded state) 2- Thermal and Environmental stability (Temperature, pH, solvent, detergents, salts.....)

34. Thermal & Environmental stability: 1. Stabilization of alpha Helix 2. Engineer structural Motifs ( like Helix N- caps) 3. Introduction of salt bridges

35. Continued... 4. Introduction of residues with higher intrinsic properties for their conformation state (e.g Ala replacement within a helix) 5. Introduction of disulfide Bridges

36. Protein Engineering- Applications Some industrial Enzyme & their commercial use

37. Applications

38. Factors which contribute to stability 1- Hydrophobicity ( hydrophobic core) 2- Electrostatic interactions: →salt bridges →hydrogen bonding →dipole interactions

39. Continued... 3- Disulfide bridges 4- Metal binding ( metal chelating site)

40. The End