1. Membrane proteins ECB Fig. 11-4

2. Membrane proteins have a variety of functions

3. Association of proteins with membranes Fig. 11-21  helix or  barrel

4.  -helix transmembranedomain Transmembrane proteins span the bilayer Hydrophobic R groups of a.a. interact with fatty acid chains

5. Nonpolar a.a. Polar a.a. Multiple transmembrane helices in one polypeptide Hydrophilic pore Membrane transporter for polar or charged molecules

6. Bleach with laser beam If protein is mobile then fluorescent signal moves back into bleached area Mobility of transmembrane proteins Recovery rate measures mobility ECB Fig. 11-36

7. Peripheral membrane proteins (associated with membrane, but not in bilayer)

8. Membrane Proteins Enzyme mechanisms Kinetic parameters of enzymes Binding sites Free energy Activation energy, enzyme function Lecture 5 (cont’d) Proteins as enzymes Proteins as membrane transporters

9. Enzyme (protein) Binding site Substrate(ligand) Non-covalentinteractions Enzymes bind substrates ECB Fig. 4-30

10. Free energy is amount of useful energy available to do work How do enzymes work? Start by considering free energy  In a chemical reaction  G =      S    heat; heat released is negative  S = entropy (randomness); increased randomness is positive Reactions occur spontaneously if  G is negative  G (Delta G) = free energy change (Reactants - Products)

11. Enzymes lower activation energy but have NO effect on  G GGGG Activationenergy Energy of reactants products Uncatalyzed reaction Catalyzed reaction ECB Fig. 3-13

12. Uncatalyzed reaction X Y Enzyme catalyzed reaction X Y Enzymes accelerate reaction rates ECB Fig. 3-26

13. Enzymes can hold substrates in positions that encourage reactions to occur Enzymes can change the ionic environment of substrates, accelerating the reaction Enzymes can put physical stress on substrates Lower activation energy How do enzymes accelerate reactions? Adapted from ECB Fig. 4-35

14. Y Solution: couple to reaction where  G - (Often hydrolysis of ATP) Thermodynamically Unfavorable Reactions (  G+)  G + X Y  G + ATPADP + P i  G - X + ATP Y + ADP + Pi +  G - Many reactions in cells have positive  G: e.g. condensation reactions (forming polymers reduces randomness so  S -,  G +)  G =      S

15. Example of coupled reaction: synthesis of sucrose ECB Panel 3-1  G values are additive

16. ADP + P i + energy ATP  G of hydrolysis = -7.3 kcal/mole (Nucleotide)

17. Binding in the active site can prevent substrate interaction Enzymes can be regulated Inhibitors can bind to active site

18. Enzymes can be regulated at sites other than the active site Example: phosphorylation Fig. 5-36 ECB 4-41

19. Membrane Proteins Proteins as membrane transporters (Ch 12 ECB) Protein Secondary Structure Channel Carrier proteins Facilitated diffusion Active transport Lecture 5 Outline Proteins as enzymes

20. Properties of a pure synthetic lipid bilayer IONS H +, Na +, HCO 3 -, K +, Ca 2+, Cl -, Mg 2+ Large, uncharged Polar molecules Amino acids, glucose, nucleotides Small Uncharged polar molecules H 2 O, glycerol, ethanol Small hydrophobic Molecules O 2, CO 2, N 2, benzene Lipid Bilayer Permeability ECB 12-2

21. Transmembrane proteins allow movement of molecules that cannot move through bilayer But it is not that simple…………… ECB 12-1

22. Charged molecules - transport influenced by concentration gradient and membrane potential (electrochemical (EC) gradient) out in Concentration gradient only Membrane impermeability results in electrical and chemical gradients across membrane Conc. Gradient with membrane potential (-) inside Electrochemicalgradient ECB 12-8

23. Ion gradients across the plasma membrane Different electrochemical gradient for each ion pH 7.2* pH 7.4* Electrical and concentration gradient can be opposite (e.g. K + )

24. - Need to get an impermeable molecule across the membrane - going WITH its electrochemical gradient - Need to get a molecule (permeable or impermeable) across the membrane going AGAINST its electrochemical gradient Solution -- specialized membrane proteins for transport functions. Transport problems faced by cells:

25. Two broad classes of transmembrane proteins A. channel protein B. carrier proteins Conformational change ECB 12-3

26. Transport can be passive or active electrochemical ECB 12-4

27. Channels - Channels - Passive transport down elecrochemical gradient Channelprotein Channel-mediateddiffusion (facilitated diffusion) Impermeable ECB 12-4

28. Channel structure ECB 11-24 Aqueous pore due to polar and charged R groups Always passive transport

29. Mechanism of K + channel selectivity

30. Slower than channels Transfer across membrane driven by conformational change in transporter Binds transported ligand - highly specific Carrier Proteins: Active transport (energy-driven) Transport against EC gradient Carrier mediated Diffusion (facilitated diffusion down EC gradient) ECB 12-7

31. Active transport - three types -uses energy to drive transport against EC gradient through carrier protein ECB 12-9

32. Antiport- move opposite directions CotransportedMolecule (against EC gradient) Down EC gradient Symport- move same direction Coupled transport ECB 12-13

33. Move glucose against its EC gradient, using the energy stored in the Na + gradient. Na-Glucose symporter ECB 12-14

34. ATP-driven pumps ATP ADP + Pi Move against EC gradient Typically move ions generating EC gradient EC gradient can then be used in coupled transport

35. Na + /K + pump in animal cells ECB 12-10

36. Cyclic transport by Na + /K + pump Phosphoryation regulates the enzyme conformation Conf. change 1 Low affinity Na binding sites High affinity K binding sites Conf. change 2 High affinity Na binding sites Low affinity K+ binding sites 3 3 2 2 2 3 NaKATPase.avi

37. Chemiosmotic coupling of pumps and cotransport H + transporters in vacuole and lysosome are similar

38. Osmosis Osmosis: movement of water from region of low solute concentration to region of high solute concentration (or high water potential to low water potential)

39. How do cells prevent osmotic swelling? ECB 12-17