Cell Biology & Molecular Biology of The Cell - PowerPoint Presentation

1. Cell Biology & Molecular Biology of The CellLecturerDr. Kamal E. M. Elkahlout, Assistant Professor of BiotechnnolgyLecture 2Protein Structure and FunctionIntroduction to biotechnology registration for Biotech MSc1

2. Protein Central DogmaProteins are large molecules that are formed as single, unbranched chains of amino acid monomers– But, proteins can be turned into branched structures by ubiquitin and other ubiquitin-like moleculesThere are 20 different amino acids commonly found in proteins• A protein’s amino acid sequence determines its three dimensional structure (conformation)– Well, sort of….A protein’s 3-dimensional structure determines its chemical function(s)– (along with a whole lot of different post-translational modifications that can alter parts of its structure and change its functions)2

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4. All amino acids have the samegeneral structure4

5. Fig. 2-14: The 20 common amino acids found in proteins. 5

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7. Basic amino acids have a positivecharge at pH 7.07

8. Acidic amino acids have a negativecharge at pH 78

9. 4 of the hydrophilic amino acids arepolar, but uncharged9

10. The remaining amino acids have hydrophobic and “special” functional groups10

11. Selenocysteine is the 21stgenetic encoded amino acid11

12. Amino acids are linked by an amide linkage, called a peptide bond, to form polypeptide chains12

13. Peptide bonds and the α carbon atoms form the linear backbone of proteins, which is a regular, repeating unit The functional groups of amino acids form “side chains” that are connected to the backbone.13

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16. Polypeptide chains are flexible, butconformationally restricted16

17. The shape of proteins is determinedthrough 4 levels of structure Primary: the linear sequence of amino acidsSecondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet)Tertiary: the overall, three-dimensional arrangement of the polypeptide chainQuaternary: the association of two or more polypeptides into a multi-subunit complexThe final, 3-dimensional, folded structure is generally one in which the free energy of the molecule is minimized17

18. Three types of weak, non-covalent bonds also constrain the folding of proteins into their energy minimized 3-D structures18

19. Hydrophobic interactions also playa role in determining protein shapeResidues with hydrophobic side chains tend to cluster in the interior of the protein molecule, avoiding contact with waterPolar side chains tend to be arranged on the outsides of proteins in contact with the aqueous medium19

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21. All of these bonds are about 30-300 times weaker than covalent bondsSo why are they important?Many weak bonds applied together can produce a large force. The stability of a protein is determined by the combined strength of many non-covalent bonds21

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23. Secondary StructureThe α-helix and the β-sheet are two regular folding patterns found in almost all proteinsWhat produces these structures and why are they so common?They result from hydrogen bonding between multiple N-H and C=O groups in the backbone.Side chains are not involved in these structures.23

24. The α-helical backbone is a rigid cylinder with the amino acid side chains protruding from its surface24

25. ▲ FIGURE 3-2 Structure of a tripeptide. Peptide bonds (yellow) link the amide nitrogen atom (blue) of one amino acid (aa) with the carbonyl carbon atom (gray) of an adjacent one in the linear polymers known as peptides or polypeptides, depending on their length. Proteins are polypeptides that have folded into a defined three-dimensional structure (conformation).The side chains, or R groups (green), extending from the carbon atoms (black) of the amino acids composing a protein largely determine its properties. At physiological pH values, the terminal amino and carboxyl groups are ionized.25

26. ▲ FIGURE 3-3 The helix, a common secondary structure in proteins. The polypeptide backbone (red) is folded into a spiral that is held in place by hydrogen bonds between backbone oxygen and hydrogen atoms. The outer surface of the helix is covered by the side-chain R groups (green).Side chains protrude from the surface of the cylinder26

27. α-helices can form very stable coiled-coil structures through hydrophobic interactions between non-polar side chains27

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29. β-sheets are found in the core ofmany proteinsβ-sheets are rigid, relatively flat and extended structures that are stabilized by hydrogen bonds between neighboring polypeptide strands29

30. Secondary structure: the beta sheetWhere do the side chains go?30

31. β-sheets can be in either a parallelor antiparallel orientation31

32. Most extracellular proteins are stabilized by covalent –S-S- cross links Disulfide bond formation is catalyzed in the ER prior to export32

33. Protein domains represent another important unit of organization33

34. Figure 3-12. A protein formed from four domains. In the Src (tyrosine kinase involved in signaling between cells in multicellular animals) protein shown, two of the domains form a protein kinaseenzyme, while the SH2 and SH3 domains (Src homolgy domain2 & 3) perform regulatory functions. (A) A ribbon model, with ATP substrate in red. (B) A spacing-filling model, with ATP substrate in red. Note that the site that binds ATP is positioned at the interface of the two domains that form the kinase. 34

35. Hierarchical Structure of ProteinsDomains are constructed from different combinations of α-helices and β-sheets at their coreEach combination is called a protein fold35

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37. Most large multi-domain proteins have evolved by recombination and joining of preexisting domains in new combinations (Domain Shuffling)Many small molecule binding sites in proteins are created at the surfaces between new combinations of domains37

38. Figure 3-18. Domain shuffling. An extensive shuffling of blocks of protein sequence (protein domains) has occurred during protein evolution. Those portions of a protein denoted by the same shape and color in this diagram are evolutionarily related. Serine proteases like chymotrypsin are formed from two domains (brown). In the three other proteases shown, which are highly regulated and more specialized, these two protease domains are connected to one or more domains homologous to domains found in epidermal growth factor (EGF; green), to a calcium-binding protein (yellow), or to a "kringle“ domain (blue) that contains three internal disulfide bridges.38

39. Large proteins often contain more than one polypeptide chainBinding between two protein surfaces generally involves sets of non-covalent bonds39

40. Figure 3-21. Two identical protein subunits binding together to form a symmetric protein dimer. The Cro repressor protein from bacteriophage lambda binds to DNA to turn off viral genes. Its two identical subunits bind head-to-head, held together by a combination of hydrophobic forces (blue) and a set of hydrogen bonds (yellow region).40

41. Figure 3-22. A protein molecule containing multiple copies of a singleprotein subunit. The enzyme neuraminidase (glycoside hydrolase nz, neuraminin acid) exists as a ring of four identical polypeptide chains. The small diagram shows how the repeated use of the same binding interaction forms the structure.41

42. Figure 3-23. A protein formed as a symmetric assembly of two different subunits. Hemoglobin is an abundant protein in red blood cells that contains two copies of a globin and two copies of b globin. Each of these four polypeptide chains contains a heme molecule (red), which is the site where oxygen (O2) is bound. Thus, each molecule of hemoglobin in the blood carries four molecules of oxygen.42

43. Some globular proteins can form long helical filaments• Globular proteins fold into a compact, ball-like shape with irregular surfaces• Example: Actin filaments form in a helical arrangement that can be the length of the cell43

44. Proteins can be subunits for theassembly of large structuresenzyme complexesribosomesProteasomes (large proteases, degrade uneeded damage proteins)filamentous structures (nuclear lamina)virusesmembranes44

45. Protein Function Some General PrinciplesAll proteins bind to other moleculesProtein binding has a high degree of specificity for its ligands (binding partners)Ligand specificity and affinity are determined by sets of weak non-covalent bonds and hydrophobic interactions.45

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47. Figure 3-38. The binding site of a protein. (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can make noncovalent bonds only with certain ligands. (B) A close-up of an actual binding site showing the hydrogen bonds and ionic interactions formed between a protein and its ligand (in this example, cyclic AMP is the bound ligand).47

48. Enzymes are highly specific catalystsEnzymes speed reactions by selectively stabilizing unstable transition states (conformations) of their ligandsThis lowers the activation energy of the reaction.48

49. The catalytic activities of many enzymes are highly regulated through small molecule binding sitesAllosteric enzymes have two or more binding sites that interact with other molecules– an active site that recognizes substrates– a regulatory site that recognizes a regulatory molecule binding of a regulatory molecule at one site on the protein causes a conformational change in the polypeptide that can switch the active site conformation “On” or “Off”.49

50. Figure 3-57. Positive regulation caused by conformational couplingbetween two distant binding sites. In this example, both glucose and moleculeX bind best to the closed conformation of a protein with two domains. Becauseboth glucose and molecule X drive the protein toward its closed conformation,each ligand helps the other to bind. Glucose and molecule X are therefore saidto bind cooperatively to the protein.50

51. How does a cell regulate proteinfunction?Cells can regulate the steady state levels of proteins through synthesis or degradation– Regulate mRNA levels by controlling transcription or mRNA stability– Control of translation of a protein’s mRNA– Targeted degradation of a protein through proteolysisChanging the activity of a protein through conformational changesChanging the location of a protein by moving it to a different part of the cell51

52. Protein structure and function can also be regulated by covalent modifications of exposed residuesN-terminal acetylation stabilizes proteins– non-acetylated proteins are degradedrapidly by proteases52

53. Acetylation and Deacetylation of Histone Tails Control Transcription ActivityDeacetylation inhibits binding of transcription factors to the TATA box, repressing gene expressionHyperacetylation of histone N-terminal tails facilitates access of general transcription factors needed for transcription initiation53

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55. A few of the chemical modifications found on functional groups on internal residues55

56. Ubiquitin is one of a family of small proteins that can be covalently linked to the ε- aminogroup of exposed lysine residuesUbiquitin covalently links its C-terminal Glycine residue to the ε-amino group of lysine through an iso-peptide bond.56

57. Polyubiquitination can target proteins for degradation in proteasomes57

58. Monoubiquitination, or Linkage of Small Ubiquitin-Like Molecules (SUMOS) Can Regulate Protein Structure and ActivityIn cells, many changes in protein binding / catalytic functions are driven by phosphorylationCells contain a large collection of protein kinases and phosphorylasesWhat amino acids are phosphorylated?58

59. Phosphorylated amino acids59

60. Protein phosphorylation and dephosphorylation play a major role in regulating enzyme activity and in driving the regulated assembly and disassembly of protein complexesAddition of a phosphate group (2 – charges) to a residue can attract + charged side chains, causing major conformation changesAttached PO4 groups can form structures that can be recognized as binding sites by other proteins.60

61. Phosphorylation / dephosphorylationProtein phosphorylation is reversible and can act as a molecular switchDephosphorylation can restore original conformation and activity of the proteinNote: In this case the dephosphorylatedform of the protein is active61

62. Other proteins bind and hydrolyze GTP to act as a molecular switch (GTP binding proteins or GTPases)Actually another form of phosphorylation/ dephosphorylationGTP binds tightly to protein, usually activating it.Protein can self-catalyze conversion from GTP to GDP.Conformational change converts protein to inactive form.62

63. 63Activation of Ras signaling causes cell growth deferentiation and survival

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65. 65GEF guanine exchange facto, GAP GTPase activating factor

66. Phosphoproteins can serve as signal integrators for a molecular switchExample: Activation of a protein requires the input of multiple signals from different parts of the cellcdk kinase (cyclin dependent kinase) – involved in cell division66

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68. Different graphical representations ofthe same protein68

69. Different graphical representations ofthe same protein69