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Transcript
Section 5 Junaid Malek, M.D. Membrane Proteins • Most membrane functions carried out by proteins, which comprise 50% of the mass of most membranes • Functions: receptors, transporters, anchors or enzymes • Associations: transmembrane, membraneassociated, lipid-linked, protein attached • We can use different tests to determine protein-membrane associations • Adding detergent - Using amphipathic molecules • Adding lipase - an enzyme that can degrade protein/lipid covalent bonds • Salt wash - washing with a high concentration of salt in water disrupts ionic and H-bond interactions Effects on MembraneAssociated Proteins Technique Result Detergent added Liberated Lipase No effect Salt wash No effect Effects on Lipid-Linked Proteins Technique Result Detergent added Liberated Lipase Liberates fragment Salt wash No effect Effects on ProteinAttached Proteins Technique Result Detergent added Liberated Lipase No effect Salt wash Liberates fragment Effects on Transmembrane Proteins Technique Result Detergent added Liberated Lipase No effect Salt wash No effect Transmembrane Proteins • Utilize specific secondary structures to bridge hydrophobic span of lipid bilayer • α-helix • β-sheet In these secondary structures, where are the side-chains located? • α-helix • Side-chains stick out from the helix • β-sheet • Side-chains alternate facing inside of the barrel and outside of the barrel Lipid Rafts • Slightly thicker regions of the cell membrane (longer FA chains) • Region of membrane specialization • Different proteins can segregate there with different function • Q: Why might selective segregation and concentration be important for membrane function? • A: They help sort proteins to the correct destination. They could concentrate signaling proteins in the same area so once activation occurs the signal can be rapidly transmitted. Membranes as a barrier to restrict molecular movement Which molecules can freely diffuse across a lipid membrane? Glycine? No Glutamate? No Methanol? Yes Water? Yes O2? Yes H+? No Cellular Transport • In order to survive and grow, cells must exchange molecules with their environment • These molecules moved across the membrane by special transport proteins • Transport can be active (requiring energy) or passive (not requiring energy) Cellular Homeostasis • Cells keep an internal ion concentration different from the external environment • This difference is crucial to cellular function, including function of nerve cells Intracellular Extracellular Na+ + Na Cl- Cl + K K+ Key Points • Sodium is the most plentiful extracellular ion. Potassium is the most plentiful intracellular ion. • Too much electrical charge cannot build up inside or outside of the cell so the amount of positive charge inside (or outside) of the cell must be balanced with an almost equal number of negatively charge • Outside the cell, the high concentration of sodium is balanced by chloride • Inside the cell, the high concentration of potassium is balanced by negatively charged organic ions (anions) Key Points • Tiny excesses of negative or positive charge can build up near the plasma membrane - this has important consequences • Uncharged molecules - concentration gradient drives passive transport The Electrochemical Gradient • Influences transport of charged molecules • Combination of the concentration gradient and the membrane potential (charge difference across the membrane) • Active transport of ions against the electrochemical gradient is crucial for maintaining the internal ion composition of cells Electrochemical Gradient • Two factors can influence ion movement: concentration of the ion and concentration of charges • If an ion like Na+ is trying to enter a cell, it is moving down it’s concentration gradient, and also from a region that is more positive to a region that is more negative. The energy released is greater than that of either concentration or charge alone. Electrochemical Gradient • If an ion like K+ is trying to exit a cell, it is moving down it’s concentration gradient, but up the charge gradient. Thus the energy released when K+ exits the cell is less than one would expect relying on concentration alone. Ion Channels • Exhibit tremendous selectivity and efficiency • Remember, structure affects function! Potassium Channel • 4 subunits positioned precisely against one another • Has a pore of defined diameter that will only allow one ion (stripped of water) to go through at a time Potassium Channel • Remember thermodynamics: Na+ or K+ can interact with 4 water molecules (favorable enthalpy). When in the pore, Na+ is smaller and can only form bonds with 2 carbonyl oxygens (enthalpically unfavorable) while K+ can form bonds with 4 carbonyl oxygens (enthalpically neutral) Where can the carbonyl oxygens in the pore come from? carbonyl oxygen can also come from peptide backbone Membrane Potential • Originates from a charge imbalance due to the movement of potassium ions • Created due to the flow of K+ through leak channels and the Na+/K+ ATPase pump, though mainly due to the K+ leak channels Membrane Potential • A charge imbalance is created and then K+ is allowed to leak in to try to balance the charge imbalance. K+ enters attracted by the negative charge inside the cell, but the concentration has a limit based on the disfavorable process of moving the ions from a region of low concentration to high. • Thus a point is reached where there is a balance between the electrical gradient and the concentration gradient of K+ so the electrochemical gradient for K+ is 0 HIV Entry Into The Cell: Membrane Insertion • gp120 binds to the chemokine receptor • This induces a change in the shape of gp120 that allows gp41 to unfold so the 3 N- and 3 C- alpha helices come apart • gp41 springs out and spears the plasma membrane of the host cell with its tip (called the fusion peptide) anchoring the HIV virus to the host cell What type of amino acids would you expect to be present in this tip? • Hydrophobic amino acids HIV Entry Into The Cell: Membrane Fusion • The pre-hairpin intermediate (gp41 “stretched out”) spontaneously rearranges back into a hairpin so that its alpha helices bundles are now close • The energy released by this favorable structural rearrangement is used to pull the two membranes together Fuzeon: HIV Fusion Inhibitor • 36AA peptide corresponding to a region from the C-terminus of gp41 • Binds to the N-terminal α-helical bundles and prevents them from binding to the real C-terminal bundles • This causes the virus-host cell complex to get stuck in the pre-hairpin intermediate • In the absence of fusion, the virus will fall off the cell fairly rapidly Prokaryotic Cell • plasma membrane, cell wall, periplasmic space, no compartments, genetic material is DNA-organized into nucleoid Eukaryotic Cell • plasma membrane surrounds cell, eukaryotic cells much more complex, organized into membrane-bound compartments called organelles, genetic material is DNA – contained in nucleus The Organelles • Mitochondria - energy generators of the cell that make ATP; surrounded by a double membrane • Endoplasmic Reticulum (ER) - a maze of interconnected spaces surrounded by a membrane serves as the site of synthesis of proteins destined for membranes The Organelles • Golgi Apparatus - stack of flattened disks of membrane that receives proteins from the ER, modifies them and directs them to other organelles, the plasma membrane or to the exterior of the cell • Lysosomes - site of degradation of macromolecules • Peroxisomes - contained environment for reactions involving hydrogen peroxide, a highly reactive molecule Virus Anatomy Cellular Transport Cellular Transport • Nucleus - transport through nuclear pores • Large enough that ions and small molecules (e.g. metabolites) can freely diffuse through them, but proteins and nucleic acids cannot • Chloroplast, ER, mitochondrion - no pore • Proteins, ions, and small molecules must be transported across the membranes Cellular Transport • Proteins move within the secretory pathway between the ER, Golgi, plasma membrane, and lysosomes • Movement between these compartments occurs inside lipid vesicles • Vesicles are loaded with cargo proteins from the lumen, or interior space, of one compartment, and discharge their cargo into a second compartment Protein Targeting • Signal sequences are portions of proteins that act as a zip code to tell the cellular machinery where the protein’s correct destination is • Signal sequences are recognized by transport receptors, which help target proteins to the correct compartment • Typical signal sequences can either be contiguous stretches of AAs or they can consist of AAs distributed throughout the protein sequence, which are close together in the folded structure of the protein Targeting Proteins to ER • ER is entrypoint for proteins going to lysosome, GA and cell membrane • Entry directed by signal sequence • Most proteins enter the ER before they are fully translated • Both water soluble and transmembrane proteins can be transferred from cytosol to ER • Once in ER, though, these proteins rarely return to cytosol Targeting of gp160 to the ER • gp160 is an integral membrane protein in that it is the precursor to gp120 and gp41 • gp160 has an N-terminal signal sequence that directs it to the ER Processing of gp160 • During protein translocation into the ER, the signal peptide is cleaved by the signal peptidase • The growing polypeptide is modified by the addition of N-linked oligosaccharides within the lumen of the ER • Proteins inside the ER help gp160 fold and promote the formation of the correct disulfide bonds Processing of gp160 • gp160 possesses ~30 potential N-linked glycosylation sites and 10 disulfide bonds • Without proper glycosylation and disulfide bond formation, the newly synthesized gp160 proteins aggregate and remain in the ER