BS332 Biomembranes and Bioenergetics Past Paper PDF

BS332: Biomembranes and Bioenergetics  BS332 Course Structure 17 lectures will be delivered by Prof Vass Bavro Dr Dima Svistunenko Any problems contact the module supervisor: Prof. Vass Bavro 2 h – revision worksho...

BS332: Biomembranes and Bioenergetics  BS332 Course Structure 17 lectures will be delivered by Prof Vass Bavro Dr Dima Svistunenko Any problems contact the module supervisor: Prof. Vass Bavro 2 h – revision workshop [email protected] Assessment: 100 percent - 3 hour Summer Exam Mark (TBC: week 33-36)  BS332 Course Structure Week Lecture No, title Lecturer VB – 2 1. Introduction to module. Membrane structure in relation to function. Biogenesis and dynamic nature of membranes. VB Wednesday 2 2. Types of lipids in biomembranes and their phase transitions. Lipid rafts. Detergents and membrane stability. VB –once! 2 3. Membrane proteins. Mechanisms of membrane protein folding and membrane insertion. Co-translational and post-translational insertion and secretion. Sec and Tat pathways. VB Then on 4. Differences between bacterial and eukaryotic membranes. Membrane transport in bacteria and organelles. 3 Mondays 5. Eukaryotic membrane synthesis, turnover and intracellular targeting. VB 3PM-5PM 3 VB 6. A recapitulation of different forms of energy, energy conservation law, energy used by biological systems, the first 3 and second laws of thermodynamics. DS 7. Introduction of state functions, ∆G, ∆H and ∆S. Discussion of negative ∆G values and the criterion for spontaneity. 4 DS 8. Definition of G° and the relationship with equilibrium constant K. Relationship between ∆G, ∆G ° and Γ (Mass 4 Action Ratio). Calculation examples. DS 9. Application of the relationship between G and K and  to the calculation of the free energy change associated with ATP hydrolysis. Data analysis using Excel spreadsheet-calculator. 5 DS 10. Hydrophilic, hydrophobic and amphipathic properties of molecules. Hydrophobic ‘attraction’. Thermodynamics as the driving force of conformational changes in proteins during folding. 5 DS 11. Redox reactions and electron transfer in biological systems. Redox potentials, standard redox potentials, relation 6 to free energy. DS 12. The Nernst equation and membrane electrical potentials. 6 DS 13. Ion channels, electrogenic pumps, resting and action potentials. 7 DS 14. Transport of ions and solutes across membranes. Passive (diffusion and facilitated diffusion) and active transport 7 processes. VB 15. Transporters and molecular motors. F1F0-ATP synthase. 7 VB 16. Chemiosmotic theory. Oxidative phosphorylation by mitochondria. 7 VB This 8list is also available in the Module Handbook on Moodle 17. Photosynthesis. Light energy transduction and photophosphorylation by chloroplasts VB https://moodle.essex.ac.uk/course/view.php?id=12353 30 Revision workshop (2 h) VB/DS How to navigate your on-line resources Your primary websites are: The BS332-6-AU-CO module info on the Module Directory: https://www1.essex.ac.uk/modules/Default.aspx?coursecode=BS332&l evel=6&period=AU&campus=CO&year=21 and the Moodle page: https://moodle.essex.ac.uk/course/view.php?id=12353 …from which you can access The Module Handbook Reading list site Announcements, if any! Sample exam paper Lectures – will be uploaded around the date and time of delivery! Recommended reading resources Bioenergetics 4 Molecular Biology of the Cell  Recommended reading resources Nicholls D.G., Bioenergetics (4th Edition); Academic Press; 4 edition (July 30, 2013); ISBN-10: 012388425X; ISBN-13: 978-0123884251 Haynie D.T., Biological Thermodynamics (2nd Edition). Cambridge University Press; 2 edition (March 10, 2008) ISBN-13: 978-0521711340; ISBN-10: 0521711347 Alberts B., Johnson A.D. Lewis J., Morgan D., Raff M., Roberts K., Walter P. Molecular Biology of the Cell (6th Edition) ISBN-13: 978-0815344322; ISBN-10: 0815344325 Madigan M.T., Matrinko, J.M., Buckley D.H, Stahl D.A., Brock T., Brock Biology of Microorganisms , Pearson; 14 edition (January 12, 2014) ISBN-13: 978-0321897398 ;ISBN-10: 0321897390 - especially – Chapter 13 – Metabolic diversity of Microogranisms pp 403-455 Atkins, P., de Paula, J. Atkins' Physical Chemistry. Oxford University Press, Oxford, 2014, 10-th edition, 1005 pp ISBN 9780199543373 Powers, Joseph M. Lecture Notes on Thermodynamics (https://www3.nd.edu/~powers/ame.20231/notes.pdf ) (It is recommended to download the PDF for convenient off-line use, DS).  Today’s lecture: Introductions to Biomembranes. What are lipids. Organisation of pro- and eukaryotic membranes. What are membranes? Main lipids forming the membrane Models of the membrane Membrane asymmetry and its significance Organisation of bacterial and eukaryotic membranes Organelles and endosymbiosis theory STAR SLIDE - DENOTES AN EXPANDED INFORMATION SLIDE!  Learning outcomes Describe the components of membranes (proteins/polysaccharides/lipids) and explain their functional roles. Describe how proteins and lipids can move within membranes. Explain why membranes are fluid; what is membrane asymmetry and how this is maintained. Explain the differences between eukaryotic and prokaryotic membranes.  What are membranes? Complex entities of lipid and protein Assembly of lipid and proteins, some can contain water and sugar Fluid dynamic structures 50% of the mass of animal membranes is lipid Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008)  What are membranes? Lipid-formed bilayers The two layers are sometimes referred to as two leaflets Impermeable to most water-soluble molecules; relatively inert Almost all functional activities are mediated by membrane proteins Some membrane proteins go across the whole membrane and are called ‘transmembrane’ proteins, others are peripheral ~30% of the genome encodes membrane proteins, but over 50% of all drugs target them! Can you think of a job for a membrane protein? Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008)  Why have membranes? Permeability barriers! Gatekeepers of the cell! Essential to life, they compartmentalize biochemical reactions Allow build-up and maintenance of gradients (ions, protons, charge, metabolites) and enable energy conversion. Surround organelles Have their own functionality Like a paper filter, membranes provide higher concentrations of reagents in enzymatic reactions – helps to drive them faster  How lipids pack into an aqueous environment is determined by their shape Cone shaped lipids form micelles Cylindrical phospholipids form bilayers The hydrophilic moieties interact with solution while the fatty acid tails bury themselves to form a water-free core Amphipaticity is KEY! Figure 10-7 Molecular Biology of the Cell (© Garland Science 2008)  The bilayer The bilayer structure based on amphiphilic phospholipids was first suggested by Gorter and Grendel (1925) based on experiments in which they extracted lipid from human erythrocytes and spread it at an air-water interface. The surface area covered by the lipid was twice that of all erythrocytes. From that they concluded that a lipid bilayer formed the plasma membrane of the erythrocyte. The idea of proteins in the membrane was introduced by Danielli and Davson (1935) – although through an erroneous “trilamellar sandwich model”. Modern understanding of the membrane - the Fluid Mosaic model - Singer and Nicolson (1972). Singer, S. J. and Nicolson, G.L. (1972)The fluid mosaic model of the structure of cell membranes. Science, 175, 720-731 Gorter, E. and Grendel, F. (1925). On bimolecular layers of lipids on the chromocytes of the blood. J. Exp. Med. 41, 439-443  Lipid plus protein = biomembrane Membrane proteins act as: - Passive diffusion gates (controlled or not) - Ion channels - Active transporters (sugars, amino-acids, protons) - Energy generators (ATPases; Photosystems) - Signalling and sensor molecules - Adhesins and receptors (immune response) - Defensive and toxic weapons Protein is not always in a minority! Bacteriorhodopsin filled purple membrane Nicholls D. G., Ferguson S, J. Bioenergetics Nature Protocols 2, 2191 - 2197 (2007) ; doi:10.1038/nprot.2007.309  Basic building blocks - lipids Ester bond formation in glycerides Fatty acid (Palmitate) - saturated Glycerol Fatty acid (Oleate) - Phosphate unsaturated Sphingosine Amide bond These are examples of condensation reactions with release of water  Diversity of membrane lipids Cells contain over a 1000 different lipid species Main membrane lipid classes in the eukaryotic cell: Glycerids and Sphingolipids Phospholipids Cholesterol derivatives Glycolipids Molecular LEGO! Sphingosine Sphingomyelin Ganglioside = Cerebroside = cerebroside + Ceramide = ceramide + sialic acid (sphingosine + fatty acid) sugar Prog Lipid Res. 2016 Apr;62:75-92. Main membrane-forming lipids  The phosphoglyceride molecule (or Glycerophospholipid) A phospholipid Variable moiety (phosphatidylcholine) The central part of the skeleton is a glycerol moiety. It is a trivalent alcohol, that is derivatised with 2 fatty acid chains plus a phosphate moiety. The composition of the lipid is important to the properties of the membrane Fatty acid tail Chain kinks affect mobility and 12-24 carbons hence membrane fluidity Saturated Unsaturated aliphatic chain aliphatic chain Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008)  Main lipids of mammalian membrane Some lipids can be charged e.g. phosphatidylserine carries a net negative electrical charge (pH dependent!) Negative Positive Neutral Neutral Neutral charge charge Figure 10-3 Molecular Biology of the Cell (© Garland Science 2008)  Cholesterol affects diffusion Up to 1 per phospholipid in eukaryotes Cholesterol orientates with the polar head group close to the polar groups on the phospholipid molecules – the rigid sterol ring interacts with the regions of the hydrocarbon chains that are closest to the polar head groups Figure 10-4&5 Molecular Biology of the Cell (© Garland Science 2008)  Cholesterol in a lipid bilayer Note! Cholesterol does NOT form a bilayer by itself! By decreasing the mobility of the first few – CH2 groups of the hydrocarbon chains of the phospholipid molecules, cholesterol makes the lipid bilayer more rigid in this region thus decreasing the permeability of the membrane to small water-soluble molecules, e.g sugars at the high concentrations found in eukaryotic plasma cholesterol prevents hydrocarbon chains from coming together and inhibits possible phase transitions into crystalline state thus affecting the membrane’s response to temperature Figure 10-5 Molecular Biology of the Cell (© Garland Science 2008)  Membrane asymmetry SM -Pphingomyosin PC – Phosphatidyl Choline PE – Phosphatidyl Ethanolamine PS - Phosphatidyl serine Crit Rev Biochem Mol Biol. 2009 Sep–Oct; 44(5): 264–277. Phosphatidyl serine (PS), which is negatively charged, is only found in the inner layer, consequently surface of inner layer is negatively charged relative to surface of outer layer. Inositol phospholipids (PI) are concentrated mostly in inner leaflet of the bilayer Glycolipids only found in outer layer – sugar groups protrude into intercellular space.  Asymmetry in the lipids is functionally significant! Phosphatidylserine (PS) is negatively charged and found on the inner leaflet – used as a marker for cell status: Red Blood Cells – important for coagulation cascade Signalling - Protein kinases (e.g. Protein kinase C) use the negative membrane to become active. PS distribution can be used to distinguish living from dead/dying cells, in apoptotic cells the phosphatidylserine randomises in the membrane and its appearance on the outside is a signal for phagocytosis of apoptotic cells Figure 10-16 Molecular Biology of the Cell (© Garland Science 2008) HUMAN RED BLOOD CELL MEMBRANE  Phospholipid translocators Plasma membrane – ER – highly symmetric & asymmetric & randomised actively maintained! Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)  Asymmetry in the lipids is functionally significant – phosphatidyl inositol signalling A signal results in phosphorylation of phosphatidylinositol (PI) This can now recruit a signalling molecule resulting in downstream activation The effects don’t have to be local, the phospholipid can diffuse Figure 10-17a Molecular Biology of the Cell (© Garland Science 2008)  Membrane asymmetry – why is it important? Apoptosis! Unequal distribution is a result of ACTIVE transport! Factors released upon activation of cell death program, such as the mitochondrial apoptogenic factor WAH1 exit the mitochondria and activate the scramblase activity. While disruption of ATP supply Crit Rev Biochem Mol Biol. 2009 Sep–Oct; 44(5): 264–277. disables the aminophospholipid translocase.  Asymmetry is not only across leaflets – lateral asymmetry creates mosaic of microdomains! Dead Alive Damaged A commonly used technique to demonstrate lateral heterogeneity in the cell membrane is fluorescence recovery after photobleaching (FRAP) or Fluorescence photobleaching recovery (FPR). We will revisit this in our next lecture – lipidic rafts Ladha, S., et al.,.(1997). J. Cell Science, 110, 1041-1050 https://www.ncbi.nlm.nih.gov/pubmed/9175700/ Basic organisation of eukaryotic vs prokaryotic membranes  Membranes in the eukaryotic cell Figure 1-30 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)  Eukaryotic membranes Membranes are dynamic and fluid structures. Lipid bilayer provides basic structure in which proteins are embedded and attached to. LIPIDS Constitute 50% mass of animal cell membranes 1 m2 of lipid bilayer contains approximately 5 x 106 lipid molecules Plasma membrane of small animal cell contains about 109 lipid molecules All lipids are amphipathic – have hydrophilic and hydrophobic regions Membranes contain different lipid types: 1. Phospholipids – most abundant! 2. Sphingolipids – contain sphingosine 3. Glycerophospholipids – contain glycerol (Revise types and structures of lipids from cell biology and biochemistry notes from last year!)  CRITICAL DIFFERENCES BETWEEN BACTERIAL AND EUKARYOTIC MEMBRANES Eukaryotic membranes possess a wide variety of phospholipids, while bacteria usually have only a few types (critically PS is very rare!). Bacteria can also form phosphorus-free membrane lipids such as ornithine lipids (OLs), sulfolipids, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), glycolipids (GLs), diacylglycerol (DAG), hopanoids (HOPs). Eukaryotic membranes do NOT contain LPS or peptidoglycan! Ornithine lipids appear to be specific to prokaryotes. Bacterial cells do NOT contain sterols – the role of sterols is played by so called hopanoid compounds. “..We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes…” Proc Natl Acad Sci U S A. 2015 Sep 22; 112(38): 11971– 11976. doi: 10.1073/pnas.1515607112  The Bacterial Cell Envelope Gram-positive 1 membrane (After the LPS Danish bacteriologist Hans Christian Gram) Gram-negative 2 (TWO!) membranes!! Cabeen MT & Jacobs-Wagner C (2005) Nature Reviews Microbiology 3, 601-610  The Gram-negative cell envelope OM lipoproteins; Braun’s LP OM proteins: (BLP)-peptidoglycan linkage TM -barrels OM outer leaflet is LPS (lipid A) Outer LPS Membrane Periplasm Inner Membrane IM proteins: IM is phospholipid (PL) in structure TM -helices Cabeen MT & Jacobs-Wagner C (2005) Nature Reviews Microbiology 3, 601-610 VN Bavro SUMMARY OF MEMBRANE ORGANISATIONS Nature Reviews Microbiology volume13, pages620–630 (2015) https://doi.org/10.1038/nrmicro3480 MICOBACTERIAL MEMBRANES – work in progress! “We found that the outer leaflet of the outer membrane contains a similar number of hydrocarbon chains as the inner leaflet composed of mycolic acids covalently linked to cell-wall arabinogalactan, thus validating the outer membrane model. Furthermore, we found that preliminary extraction with reverse micelles permitted the subsequent complete extraction of inner membrane lipids with chloroform–methanol–water, revealing that one-half of hydrocarbon chains in this membrane are contributed by an unusual lipid, diacyl phosphatidylinositol dimannoside (Ac2PIM2). The inner leaflet of this membrane likely is composed nearly entirely of this lipid.” Ritu Bansal-Mutalik and Hiroshi Nikaido PNAS April 1, 2014 111 (13) 4958-4963; https://doi.org/10.1073/pnas.1403078111 Origin of eukaryotic organelles  Endosymbiosis - mitochondria Figure 1-34 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)  Endosymbiosis - chloroplasts Figure 1-36 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)  Summary Biological membranes are a bi-layers (double layers) of phospholipids with embedded proteins Membranes are dynamic semi-fluid assemblies Main membrane lipid classes in eukaryotic cells: Phospholipids Cholesterol Glycolipids Inner and outer layer compositions can be different! Asymmetry is functionally significant! Phospholipids can be involved in signalling. Lipid rafts can form functional domains! Prokaryotic cells and eukaryotic cells have distinct membrane compositions. Cholesterol is a marker of animal cells. Lipopolysaccharides (LPS) forms the outer leaflet of the Gram-negative bacterial membrane. Prokaryotic cells generally lack membrane organelles. Mitochondria and plastids have likely originated from endosymbiotic bacteria.  Recommended additional reading There Is No Simple Model of the Plasma Membrane Organization. Front Cell Dev Biol. 2016 Sep 29;4:106. eCollection 2016. https://www.ncbi.nlm.nih.gov/pubmed/27747212 Bacterial membrane lipids: diversity in structures and pathways FEMS Microbiology Reviews, Volume 40, Issue 1, 1 January 2016, Pages 133–159, https://doi.org/10.1093/femsre/fuv008 Three-Dimensional Distribution of Phospholipids in Gram-Negative Bacteria Biochemistry 2016, 55, 34, 4742-4747 https://pubs.acs.org/doi/10.1021/acs.biochem.6b00541