Protein Engineering BME 128

Transcription

1 Protein Engineering BME Introduc5on 2. Food, phones, laptops, etc. 3. A?endance 4. Review of syllabus, office hours, DRC 5. Review of reading material, assignments, midterms, grading, etc. 6. BME 128 is a design course: Design Project (modeled strongly aper BME 177 stem cell engineering) *APPLY KNOWLEDGE FROM CLASS TO A NEW IDEA* 7. Let s get to work! 1

2 What is Protein Engineering? Design and construct of new proteins or enzymes with novel func5ons - Z. Galzie 1991 Biochemical Educa.on Design of new enzymes with new or desirable func5ons - Turanli- Yildiz et al., 2012 Procedures by which protein structure and func5on are changed or created in vitro by altering exis5ng or synthesizing new structural genes that direct the synthesis of proteins with sought- aper proper5es. - Reference.MD Muta5ng the gene of an exis5ng protein in an a?empt to alter its func5on in a predictable way Process of developing useful or valuable proteins - Wikipedia 2

3 Francis Crick s Central Dogma of Biology (1958) 3

4 Proteins%are%extremely%versa6le%biomolecules% For example Defense antibodies and complement proteins Movement contractile proteins in muscles Enzymes catalysts that speed up chemical reactions Signaling hormones and their receptors Structure mechanical support Transport membrane proteins that allow specific substances in and out of cells (it seems too narrow to restrict Transport Proteins to membrane proteins - there are more than channels!) 4

5 Proteins are made of amino acids Side chain Alpha carbon Amino group Carboxyl group 5

6 Amino acids have mul5ple ioniza5on states 6

7 Amino Acids Memorize all structures! Memorize all 1- and 3- le5er abbrevia8ons Know if side chain will be be charged at neutral ph 7

8 Small Hydrophobic%Amino%Acids% 8

9 Hydrophobic%Amino%Acids% 9

10 Aroma5c Hydrophobic%Amino%Acids% 10

11 Polar%Amino%Acids% Aroma5c 11

12 Polar%Amino%Acids% Nucleophilic Can form disulfide bonds discussed later in lecture 12

13 Acidic Nega6vely%charged%amino%acids% 13

14 Basic Posi6vely%charged%amino%acids% His5dine pka ~ 6 ph sensors in proteins 14

15 Ques5on: What is net charge of this pep5de at ph 7? Ala- His- Val 15

16 (Glycerol) (Pep5de backbone carbonyl) 16

17 How do amino acids compose a protein? 17

18 Pep5de bond forma5on 18

19 Pep5de bonds are planar R1 R2 No5ce shorter length bond: Due to resonance with carbonyl R1 Note: 1 Å (Angstrom) = 0.1 nm R2 19

20 Planarity of Peptide Linkages 20

21 Rotatable Bonds Enable Structural Versatility Steric clashes limit possible phi and psi angles: Ramachandran Plots:! vs. " 21

22 Hierarchical Levels of Protein Structure (Description) 22

23 Protein)Secondary)Structures:)The)alpha)helix) Amino terminus Carboxy terminus 3.6 residues per turn 5.4 Å (Angstroms) per turn Pep5de backbone carbonyl and amide hydrogen form hydrogen bond Helices have dipole moments due to direc5onality of hydrogen bond (in general amino terminus = + and carboxy terminus = - 23

24 Ques5ons: a. Why is proline rarely found in helices? b. What kinds of interac5ons may be stabilizing this helix? Ile - Asn - Arg - Ala - Leu - Asp - Arg Asp 24

25 Parallel)and)an-?parallel)beta)sheets) Pep5de backbone carbonyl and amide hydrogen form hydrogen bond 25

26 Mixed $-Sheets Note the $-twist in the sheet shown above! 26

27 Secondary Structure III: Turns and Loops Inbetween #-helices and $-strand segments there are often further regular, and rigid, segments - there are several types of recurring turn structures. Other connecting regions are (potentially) flexible loops. These used to be referred to as random coil, or coil segments. Careful! Most current bioinformatics tools do not distinguish between well-defined, rigid turns and flexible regions - e.g. in most secondary structure predictions, there are only three categories (#; $; c (= non-# and non-$)) 27

28 28

29 What are the forces behind protein folding? 29

30 The%hydrophobic%effect% Water%is%the%solvent%of%life!% 30

31 Protein%folding%is%largely%driven%by%hydrophobic%collapse% ΔG = ΔH TΔS 31

32 Designing the hydrophobic core Barnase is a ribonuclease from bacteria Bacillus Assay: If barnase is expressed in the absence of its inhibitor barstar, the protein will degrade RNA in the cell and thus kill the cell The assay is sensitivity enough to detect a mutant protein with > 0.2% of the activity of wild type Randomly mutate 12 of the 13 core residues to other hydrophobic residues 23% of all mutants retained enzymatic activity Hydrophobicity is a sufficient criterion for constructing a core that is capable of supporting enzymatic activity Axe et al. PNAS 93, 5590, (1996) barnase 32

33 Buried water There are many packing defects and cavities in the protein core Some cavities contain water molecules Hubbard et al., Protein Eng 7, 613 (1994). Williams et al., Protein Sci 3, 1224 (1994) Hubbard & Argos, Curr Op in Biotech 6, 375 (1995) Bound water is close to energy neutral entropic cost of immobilizing a water molecule ~ 2 kcal/mol at 300 K All amino acids (even hydrophobic residues) contain polar atoms Buried polar atoms must be H-bonded Water can satisfy the H-bonding needs of turn/loop/coil residues Distribution of bound water varies with secondary structure 33

34 Protein Engineering and the protein core Ques5on: Stabilize or destabilize? Subs5tu5ng a small hydrophobic residue with a larger hydrophobic residue could: Displacing a water molecule in the protein core could: 34

35 Protein Engineering and the protein core Ques5on: Stabilize or destabilize? Subs5tu5ng a small hydrophobic residue with a larger hydrophobic residue could: stabilize it by filling in an unfilled hydrophobic pocket destabilize it by inducing a volume change and distor5ng the overall structure Displacing a water molecule in the protein core could: It depends what is displacing the water molecule: stabilize it by forming a stronger hydrogen bond destabilize it by exposing polar atoms in the hydrophobic core 35

36 Non- covalent interac5ons among biomolecules in aqueous solvent 36

37 Name two amino acids whose side chains would - Form a hydrogen bond with a pep8de backbone carbonyl group Form an a5rac8ve ionic interac8on Form a repulsive ionic interac8on Form a hydrophobic interac8on Form a pi- stacking hydrophobic interac8on Form a covalent bond 37

38 Protein sequence determines structure and func5on Ribonuclease experiment, 1950s Theore5cally 8 Cys residues could make 4 disulfide bonds in 105 different ways Experimentally shown to be true: disulfides allowed to form in presence of denaturing solu5on 38

39 Hierarchical Levels of Protein Structure (Description) 39

40 Tertiary Structure: Different Representations Ball- and- s5ck view Ribbon view Cartoon view Surface view 40

41 Quaternary Structure: Oligomeric arrangement 41

42 Post- Transla5onal Modifica5on (PTM) Binding to co- factors: e.g. heme, metal ions Phosphoryla5on Glycosyla5on Disulfide bonds Prenyla5on / Fa?y acid acyla5on Covalent modifica5on to a C- terminal Cys residue Trunca5on / Protease cleavage Ubiqui5na5on / SUMOyla5on Acyla5on / methyla5on / Protein splicing OPen used for signaling, localiza5on, stability, metabolic regula5on h?p://themedicalbiochemistrypage.org/protein- modifica5ons.html 42

43 PTM: binding to co- factors e.g. metal ions (Zn 2+ in Alcohol Dehydrogenase) 43

44 PTM: phosphoryla5on and glycosyla5on PTM-change is primarily in: charge PTM-change is primarily in: (partial) charge molecular mass Post-translational modifications increase the number of letters in amino acid alphabet and lead to a combinatorial explosion in both database search and de novo approaches. Phosphoryla5on: Ser, Thr, Tyr N- Glycosyla5on: Asn O- Glycosyla5on: Ser, Thr 44

45 PTM: Disulfide bond forma5on Cys-Cys (Disulfide Bridges) 45

46 Disulfide bond Disulfide bond is a covalent bond formed between two cysteine side chains with the bond energy of ~ 70 kcal/mol There are strict structural requirements for ideal disulfide geometry Creighton, BioEssays 8, 57 (1988) Both isomers (right-handed and left-handed) are observed in natural proteins Particularly important in small proteins that lack genuine hydrophobic cores Protein disulfide isomerase (PDI) catalyzes internal disulfide exchange and helps correct wrong disulfide bonds that may form during folding 46

47 Removing native disulfides Removing disulfide bonds usually destabilizes the protein HEW lysozyme has three disulfides. Removing these disulfides destabilizes the protein and reduces the melting temperature by 25 C Cooper et al, JMB 225, 939 (1992) Removing disulfides in interleukin-4 significantly disrupts the integrity of its hydrophobic core Vaz et al, Protein Sci 15, 33 (2006) 47

48 Protein Engineering and Disulfide Bonds Engineering disulfide bonds into proteins can stabilize them Secretory pathway (eukaryo5c cells) = oxida5ve environment Periplasm (bacterial cells) = oxida5ve environment Cytoplasm = reducing environment Disulfide bonds can be reduced by addi5on of a reducing agent that contains thiols (e.g. BME, DTT): 48

49 Overall: Considera5ons in Protein Engineering Changes in an amino acid can affect a protein s - secondary, ter5ary, and quaternary structure - stability - solubility - func5on Can we use this informa5on to engineer useful or valuable proteins (- Wikipedia)? 49

50 Future classes Bioinforma5cs and compu5ng to predict or assess protein structure and/or func5on Prac5cal considera5ons for making a protein in the lab and assessing its structural integrity Ra5onal design and muta5on of a protein Directed evolu5on / random muta5on of a protein Case studies: hormones, an5bodies, vaccines, biomaterials 50

51 Next class: Memorize all 20 amino acids, 1- and 3- le?er abbrevia5ons Understand chemical proper5es of side chains Know how to make a pep5de bond Bring laptop into next 2 classes: Bioinforma5cs servers: protein analyses PyMOL: protein structure visualiza5on HW#1 due Tuesday: Prep for PyMOL class 51