Chapter 3-II Protein Structure and Function

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1 Chapter 3-II Protein Structure and Function GBME, SKKU Molecular & Cell Biology H.F.K.

2 Active site of the enzyme trypsin. Enzymes (proteins or RNAs) catalyze making or breaking substrate covalent bonds. (a) Trypsin (serine protease) active site: Substrate-binding pocket binds specific substrate Catalytic site contains side chains of the catalytic triad Ser-195, Asp-102, and His-57 that breaks peptide bonds. In some enzymes, the catalytic and substrate-binding sites overlap; in others, the two regions are structurally distinct.

3 Schematic model of an enzyme s reaction mechanism.

4 Free-energy reaction profiles of uncatalyzed and multistep enzyme-catalyzed reactions.

5 The real physical binding site and pocket! Substrate binding in the active site of trypsin-like serine proteases. (a) Trypsin active site: Substrate forms a two-stranded β sheet with trypsin s substrate-binding site. Trypsin binding site pocket binds substrate arginine (R 3 ) side chain Enzyme Asp-189 negative charge stabilizes substrate Arg positively charged guanidinium group. Binding aligns the substrate arginine peptide bond for hydrolysis catalyzed by the enzyme s active-site catalytic triad (side chains of Ser-195, His-57, and Asp-102). (b) Binding pocket substrate specificity:

6 Binding pockets Trypsin binds substrate (+) charged arginine and lysine side chains. Chymotrypsin binds large, hydrophobic side chains such as phenylalanine. Elastase binds small side chains such as glycine and alanine.

7 How does the serine protease work?

8 Mechanism of serine protease mediated hydrolysis of peptide bonds (a) ES complex: Ser-195 hydroxyl oxygen attacks the carbonyl carbon of the substrate s targeted peptide bond (yellow). Free electron

9 5 4 4: No further attack (b) Tetrahedral intermediate transition state: enzyme s oxyanion hole stabilizes substrate oxygen negative charge.

10 Oxyanion hole Electron acceptors An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonated oxygen or alkoxide.

11 5 4 Same figure (b) Tetrahedral intermediate transition state: enzyme s oxyanion hole stabilizes substrate oxygen negative charge.

12 6 What s next? (c) Peptide bond broken by additional electron movements: release of one of the NH 2 P 2 reaction products formation of the acyl enzyme (ESʹ complex)

13 7 8 His needs His friend to share the electron! But O is a bad friend to attack the C! [If the ph is too low: the His-57 side chain is protonated and cannot participate in catalysis.] (d) Solvent water oxygen attacks the acyl enzyme carbonyl carbon.

14 Attack 9 (e) Formation of a second tetrahedral intermediate

15 10 (f) Additional electron movements: break the Ser-195 substrate bond (formation of the EP complex) (inset) His-57 side chain held in the proper orientation by hydrogen bonding to the Asp-102 side chain facilitates catalysis by withdrawing and donating protons throughout the reaction release the P 1 COOH reaction product

16 Overall reaction

17 The ph dependence of enzyme activity. Think the first step with His residue pka = ~6.8 Trypsin protease Cytosol: ph ~7.2 Then? Protonated or Not?

18 Assembly of enzymes into efficient multienzyme complexes. Coupling by a scaffold protein overcomes slow substrate diffusion in a metabolic pathway Some enzymes are fused at the genetic level

19 Protein Structure and Function 3.4 Regulating Protein Function Proteins may be regulated at the level of protein synthesis, protein degradation, or through noncovalent or covalent in teractions. Proteins marked for destruction with a polyubiquitin tag by ubiquitin ligases are degraded in proteasomes. Several allosteric mechanisms act as switches, reversibly tu rning protein activity on and off. Higher-order regulation includes the intracellular compart mentation of proteins.

20 Ubiquitin- and proteasome-mediated proteolysis.

21 Ubiquitination Enzyme + regulatory subunit Degradation Active enzyme

22 Hemoglobin binds oxygen cooperatively. Tetrameric hemoglobin: four oxygen-binding sites Saturation all four sites loaded with oxygen. P 50 - po 2 at which half the oxygen-binding sites are occupied. Large change in oxygen bound over a small range of po 2 values permits efficient unloading of oxygen in peripheral tissues such as muscle. Sigmoidal curve indicative of cooperative binding binding of one oxygen molecule allosterically (cooperatively) influences the binding of subsequent oxygens

23 Conformational changes induced by Ca 2+ binding to calmodulin. Ca 2+ binding changes calmodulin conformation hydrophobic side chains become more exposed to solvent Ca 2+ /calmodulin complex wraps around conserved sequences in exposed helices of various target proteins, altering their activity.

24 The GTPase switch.

25 Regulation of protein activity by phosphorylation and dephosphorylation. Kinases: transfer terminal phosphate group from ATP to specific S/T or Y OH groups activate some proteins, inactivate other types of protein Most kinases can phosphorylate multiple different target proteins. Phosphatases: hydrolyze phosphate group off protein inactivates some proteins, activates other types of proteins

26 Practical protein works learned by text 나중에실험도해보시는것이

27 Protein Structure and Function 3.5 Purifying, Detecting, and Characterizing Proteins Proteins can be isolated from other cell componen ts on the basis of a variety of physical and chemical properties. Proteins can be detected and quantified by various assays and specific antibody recognition.

28 We need cells How to separate the specific types of cells? Think the difference between cells

29 Centrifugation techniques separate particles that differ in mass or density

30

31 Centrifugation techniques separate particles that differ in mass or density Max. 80,000 rpm! Boeing 747: 3,854 rpm

32 New tech. to cell separation

33 Continuous Separation of Micro-Particles by Size in a Curved Sheath Flow

34 Homogenization In cell biology or molecular biology research, homogenization is a process whereby a biological sample is brought to a state such that all fractions of the sample are equal in composition. French press

35 Sonication! Cells Sonication is the act of applying sound energy to agitate particles in a sample, for various purposes. Ultrasonic frequencies (>20 khz) are usually used, leading to the process also being known as ultrasonication or ultrasonication.

36 Think the protein properties Weight Charge Structure Which information is easy to use?

37 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins primarily on the basis of their masses. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS)

38 How does SDS work? sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS) SDS detergent for denaturation of the protein + DTT or beta-mee SDS coats the protein with a uniform negative charge. (1.4g SDS / 1g protein)

39 SDS-PAGE

40 Think the protein properties Weight Charge Structure

41 Isoelectric focusing Isoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their isoelectric point (pi). The pi is the ph at which a protein has no net charge and thus, does not migrate further in an electric field.

42 Two-dimensional gel electrophoresis separates proteins on the basis of charge and mass. Two steps! Step 1: Isoelectric focusing Step 2: SDS-PAGE separates proteins in gel based on molecular weight.

43 Two-dimensional gel electrophoresis separates proteins on the basis of charge and mass.

44 Find the difference!

45 Then what will you do?

46 How to detect the specific protein?

47 Antibody-antigen specificity + SDS-PAGE!

48 Western blotting

49 Western blotting

50 To know the sequence!

51 Find the difference! Get this protein

52 LC-MS? Mass spectrometry (MS) is an analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio. Manhattan project

53 Molecular mass can be determined by matrixassisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry. Step 1: Pulses of laser light ionize a protein or peptide mixture that is absorbed on a metal target (matrix-assisted). Step 2 3: An electric field accelerates the ions in the sample toward the detector. The time it takes an ion to reach the detector is proportional to the square root of the mass-to-charge (m/z) ratio. Results: Smaller ions with the same charge move faster (shorter time to the detector).

54 Maldi-TOF video!

55 Fingerprinting Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF.

56 Molecular mass of proteins and peptides can be determined by electrospray ionization ion-trap mass spectrometry. Top panel: Mass spectrum of a mixture of three major and several minor peptides from the mouse H-2 class I histocompatibility antigen Q10 α chain. The MS/MS spectrum (product-ion spectrum) provides detailed structural information, including peptide sequence information. peptide sequence (FIIVGYVDDTQFVR) deduction from the varying sizes of the product ions, understanding that peptide bonds are often broken in such experiments, known m/z values for individual amino acid fragments, and database information

57 Combining with LC

58 Three commonly used liquid chromatographic techniques separate proteins on the basis of mass, charge, or affinity for a specific binding partner. Instead of SDS-PAGE Column porous beads Column beads to which a specific antibody is covalently attached Columns beads with either a positive charge (shown here) or a negative charge

59 LC-MS/MS

60 LC-MS/MS is used to identify the proteins in a complex biological sample.

61 LC-MS/MS identification of organelle proteins: Think the steps to identify one protein expressed in a specific organelle.

62 Protein Structure and Function 3.6 Proteomics Proteomics systematic study of abundance, modificati ons, interactions, localization, and functions of all or su bsets of proteins in whole-organism, tissue, cellular, and subcellular biological systems. Methods used for proteomic analyses include 2D gel ele ctrophoresis, density-gradient centrifugation, and mass spectrometry. Results reveal various types of proteomes.

63 Next class Culturing and visualizing cells

64 Discussion with friends Please explain the reason why low ph inactivates trypsin protease based on enzyme reaction. See the most active phs of lysosomal hydrolase and chymotrypsin. How can you explain the inverted U-shape activity of each enzyme. If one protein was ubiquitinated, what kind of protein band change do you see on the SDS-PAGE? Find one paper in Pubmed site, Synaptic protein degradation underlies destabilization of retrieved fear memory.' Please explain the graph with allosteric mechanisms

65 Pubmed site search

66 참고

67 pka of amino acids & pi

68 Histidine pka & pi 1 3 2

69 A.As pka values Amino acid pka 1 pka 2 pka 3 pi Glycine a a a Alanine Valine Leucine Isoleucine Methionine Proline Phenylalanine Tryptophan Asparagine Glutamine Serine Threonine Tyrosine Cysteine Aspartic acid Glutamic acid Lysine Arginine Histidine

70 Disordered protein

71 Intrinsically disordered proteins: mechanisms of binding to well-ordered proteins and identification based on hydrophobicity and net charge. Conformational selection (top pathway): disordered protein (PUMA) transiently adopts its bound state structure, which then binds to the well-ordered binding partner (MLC1). Induced fit (bottom pathway): disordered protein begins to bind to the well-ordered partner and then is induced to form the ordered conformation by completing binding interactions. >30 percent of eukaryotic proteins are predicted to have at least one disordered segment of 50 or more consecutive residues.

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