Membrane Structure - BCH210H - Fall 2024 - PDF

MEMBRANE STRUCTURE INSANE IN THE MEMBRANE… Sian Patterson, Ph.D. Associate Professor, Teaching Stream  Learning Objectives By the end of this lecture, students should be able to: Review the composition and roles of biological membranes and how the diverse chemic...

MEMBRANE STRUCTURE INSANE IN THE MEMBRANE… Sian Patterson, Ph.D. Associate Professor, Teaching Stream  Learning Objectives By the end of this lecture, students should be able to: Review the composition and roles of biological membranes and how the diverse chemical structures of molecules contribute to its formation. Explain how fluorescence microscopy can be used to visualize the movement of molecules in a membrane and why membrane fluidity is important for movement. Define the unique properties of integral membrane proteins and predict the topology of a membrane protein based on the amino acids present. Describe the role of detergents and salt when purifying a membrane protein. 2  with localization helps COURSES Essential Membrane Functions Act as a barrier regulating the import and export of essential molecules. Compartmentalization of specialized processes increases cellular efficiency. The cell membrane modulates cell-cell recognition due to the presence of glycoproteins and glycolipids. sugar + Signaling across the membrane is mediated by proteins and lipids. play role in signaling a The structure of a membrane is key for its function... 3 an  between eroups non-covalent 8 ! has huge hydrophobic ↑ of water entropy portions membrane entropy ↓ of 4  Membrane Properties Biological membranes are composed of lipid bilayers that are outer) impermeable to polar or charged molecules. 2 leaflets (inner hydrophillic + hydrophobic The hydrophobic effect drives membrane formation of amphipathic molecules that assemble due to non-covalent interactions. diff in inner & outer Membranes are asymmetric and composed of lipids and proteins,. which may or may not have carbohydrates covalently bound. PTMS Membrane proteins play key roles in the transport of molecules and transduction of information across the membrane. 5  Membrane Fluidity is Important Proteins carry out the movement membrane video of molecules across a membrane. Signals may also be transmitted across a membrane. Conformational change is important for mediating these proteins help to move processes. soluble and/or membrane things across Lipids and cholesterol also play a ↳ need to change conformation role in membrane fluidity. 6 Help it to be fluid  The Fluid Mosaic Model Proposed in the early 1970s by Singer and Nicolson. ‘Fluid' – membrane components can move quite rapidly in the plane of the membrane. ‘Mosaic’ – diverse mixture of lipids, embedded and peripheral proteins, and molecules carbohydrates on the surface. mixture of Recent modifications: the movement of lipid and membrane proteins differs significant in samples of pure lipids vs. biological membranes. membrane 7 · restricted to area of an in solution movement is completly fluid Measuring Lipid Dynamics in Membranes How fast do lipids (and proteins) diffuse laterally in a membrane? OUTER lateral diffusion ? changes spots with lipids next to it INNER 8 Fluorescence Recovery After Photobleaching (FRAP) 2. Use a high-intensity laser 1. Label cell to bleach the fluorophore in 3. Measure the mobility surface molecule. a small part of the of other molecules into membrane. the affected region. bleach small part of j phrorotores regain intesity Cone side gets labled Rate = Fluorescence time 9  fluorecently tagged protein ↑ molecule recovery Voet & Voet Guided Exploration 10 · can tell weather they restricted or more freely Clatterally) SINGLE MOLECULE TRACKIN lipid raft stay here molecules - > - The to area ~ · 11  Measuring Membrane Dynamics Based on Fluorescence The recovery of lipids and membrane proteins using FRAP demonstrates their mobility in the plane of the membrane. Lipids may move very quickly (~ 1 µm/s). Single-molecule tracking fluorescence microscopy is used to monitor the movement of fluorescent molecules throughout the membrane. Some proteins or lipids may spend more time interacting within certain regions of the membrane, such as lipid rafts. The lateral diffusion of proteins may also depend on interactions with other proteins (eg. cytoskeleton) or extracellular components. inside outside 12 lipids · can track either proteins or  denatures protein SDS- > uniform charge ; lyses the cell , FRAP cannot monitor : no > crosslinker ↓ rate - recovery - covalently link proteins 13  Biological Membranes are Asymmetric The two leaflets of the membrane have very different lipid and protein compositions. PTM The addition of sugars to lipids and proteins is a form of post-translational modification mediated by enzymes and is important for membrane insertion and cell recognition. very polar are headgroups energetically un favorable ↑ The movement of lipids from one leaflet to the other is very slow. lipid movement Flip-flow diffusion of polar/charged groups across the hydrophobic membrane interior is energetically unfavorable. 14 Enzymes Mediate Membrane Asymmetry Not very slow spontenouse , proteins mediate the movement ATP - dependent 2 ATP - independent undo flipase @ flopase -in out no ATP , less specifi translocase - > even tein lipid out 15 Conc 2 categories. : > ligated molecules together ligase -  Membrane Asymmetry different distributio Enzymes assist with the heterogeneous distribution of lipids and proteins on either side of the lipid bilayer. ATP hydrolysis of flippases and floppases helps drive the movement of lipids from one membrane to the other. Scramblases move all lipids down their concentration gradient producing a symmetrical membrane. glycoproteinlycolipid formation Enzymes catalyze the addition of oligosaccharides to proteins and lipids on the extracellular surface. The synthesis of membrane proteins in the ER ensures their proper orientation. 16  LIPIDS different Lipids diverse , structures Class of molecules involved in providing structural support for cells and organelles, storage of carbons for energy, and can play a role in information transduction and signalling. Defined by their physical properties: ↓ solubility in water; ↑ solubility in non-polar solvents The physical properties arise from the presence of diverse chemical structures and functional groups Hydrophobic (only non-polar) OR amphipathic (polar and non-polar) never polar ! 17 Lipids are chemically diverse but their structure dictates their functionimportant N+ O O- P - O polar or formation hydrophobic O O O O O SDS ↑ 18  Lipid Self-Assembly and Bilayer Formation Lipids spontaneously aggregate in water to bury their hydrophobic groups while polar groups interact with water. Non-covalent forces (Hydrophobic effect, H bonds, VdWs packing) drive assembly. The structure that forms is based on the structure of lipids and their chemical interactions. non-covalent lipid tails structures depends on (liposome) ↓Con). ↑ Conc. & Made in lad 19 cell · fake  Biological Lipids Lipids metabolism STORAGE STRUCTURAL SIGNALLING Triacylglycerides Sterols Omegas Eicosanoids tail Phospholipids Sphingolipids Glycolipids Sterols Fused rings OH Functional Functional group group 20  air carbonic yearbor ↑ Fatty Acids tails P terminal methyl 2-22 carbons pKa ~4.8 l physiologica ↑ ampipathic property Fatty acyl chains are either saturated (shown) or unsaturated (double bonds). delta Double bonds are usually numbered relative to the carboxylic acid but can also be numbered relative to the terminal methyl group for omega (ω) fatty acids. count backwards They may either be free fatty acids (FFAs) or bound to a head group or backbone via ester bonds. 21  Fatty Acid Nomenclature Carbon skeleton # C : # of double bonds Eg. 18:0 bond # of double , # carbons : 29 Systematic namecarboxylic acid n-Octadecanoic acid - → 18 n-indicates “normal” (no doble bonds) kink a crates ↑ ↑ Monounsaturated: 1 C=C Cis-∆9-octadecenoate Poly (PUFA): more than 1 C=C 18:1 (ω9) 22  Omega Fatty Acids in Human Health · location of double bonds Omega-3 and omega-6 fatty acids are polyunsaturated fatty acids. Humans cannot synthesize these omega fatty acids à “essential”. Must be obtained from our diet. S 22 : 6 ↑ W3 3 omega WG 18 : Z 19 , 12 23 What are good and bad fats? acid good : Omega fatty - essential fatty acids I cannot be created bad : saturated fatty ands bad to consume - & trans fats covalent interactions clots of not - no kinks 24 Saturated vs. Non-saturated Chains A - oic acid = HA oate = 25  Chain Saturation and Membrane Fluidity form many M I non-covalent N intraction M M E W ↓+ tuidit w M N ↓non-corelent interactions ↓ fluidity ↑ melting temperature ↑ fluidity ↓ melting temperature ↳proteins 26 can go throug conformation changes  Phase Transitions lipids pure The melting of membrane lipids TM is a phase transition from a gel- like solid phase to the liquid crystalline phase. V The melting temperature (Tm) increase is an index of membrane /look at pick Before transition fluidity. Pure lipid samples have sharp, well-defined transition temperatures, while native more around membranes have broad peaks. Copyright © 2014 by Nelson Education Ltd. 27  brake apars hard to not "good" fat ; tails aggregate ; Triglyceride : a Tri-acyl-glycerols (TAGs) attached to - tails glycerol triacylglycerol Efficient, unlimited energy reserve Adipocyte (fat cell) of reduced carbon chains. stort in atipose tissue Dehydrated storage form primarily in adipocyte cells. 3 fatty acids are bound to a glycerol backbone through ester linkages. very Fatty acyl tails may also contain hydrophobic packe into small double bonds creating mixed space ↳ not in membranc triglycerides. Copyright © 2014 by Nelson Education Ltd. 28  How would the composition of lipids and TAGs differ between olive oil, butter and animal fat? = bonds olive oil many : : less double bonds butter animal fats : more saturated ↳ triglycerides 29  beX could a functio on ~ Membrane Lipid Diversity Amphipathic molecules are made by vartup a n at group attaching fatty acyl chains to polar (OH, sugar, phosphate) head groups. Glycero-phospho-lipids : → Sphingolipids : Variability exists in the structure of fatty acid 2 tails OpenStax Biology tails and polar head groups. hydrophobic · H3C Cholesterol is the most common steroid CH3 found in membranes. CH3 rigd , always HO 30 very this structu  Glycerophospholipids vs. Sphingolipids can have different charges X = Polar head groups that contribute to variability and charge. Examples of glycerophospholipids are Phosphatidylethanolamine (PE) and PS (-Serine, -ve charge), while sphingomyelin and gangliosides (sugar head groups) are examples of sphingosines. 31  H3C CH3 3. Cholesterol CH3 HO in either Can be metabolized to other hormones leaflet (cortisol, testosterone etc.) and bile salts, needed for dietary lipid absorption. Can form complexes with sphingolipids, glycolipids and some lipid-anchored proteins to form lipid rafts. Lipid rafts help moderate membrane fluidity and play a role in signal transduction. Modulates membrane fluidity. distrupts packing 32  PROTEINS outside membrane Peripheral vs. Integral Membrane Proteins associatedwith eithe heatlet b e ed c d a Created with biorender.com a, b = peripheral membrane proteins c, d = Integral membrane proteins e = lipid anchored proteins 33  1. Peripheral Membrane Proteins Adhere to the surface of lipid membranes or integral membrane proteins through non-covalent interactions. chead groupsetc. Can be removed using milder conditions (increasing salt, change in pH). Copyright © 2014 by Nelson Education Ltd. 34  completly across) 2. Integral Membrane Proteins Completely span the membrane (ie. transmembrane segments, TMs). Require harsh conditions for purification (detergents or organic solvents). ↑need to * * * * distrupt the membrane Copyright © 2014 by Nelson Education Ltd. 35  3. Lipid Anchored Proteins Also require harsh detergents or solvents transmembrane for membrane removal. segment Lipid chains are covalently attached to amino acid functional groups and side cys chains. Copyright © 2014 by N-myristoylation or S-palmitoylation are Nelson Education Ltd. examples of: GPI-anchored proteins are covalently linked through a sugar chain and lipid outside anchor (GPI = glycosyl saysA of cell) phosphatidylinositol) 36  Membrane Protein Purification Studying membrane proteins requires extra Peripheral consideration for purification and analysis. Some membrane proteins can be removed Integral using milder conditions that disrupt non- covalent interactions, while others need detergents or organic solvents to replace the lipids and disrupt hydrophobic Cisolate & Peripheral separate from interactions. membrace Detergents create micelles around d hydrophobic regions to solubilize create miscel membrane proteins. Proteins can then be purified and analyzed. 37  Studying Membrane Proteins ampipathic properties Detergents are amphipathic molecules that can help form micelles around the hydrophobic regions of a membrane protein, helping with solubilization. The critical micelle concentration (CMC) is the concentration at which the detergent spontaneously forms stable micelle structures. preserve structure Some detergents are milder (digitonin, TX-100, Tween 20) and preserve protein structure, while others are harsher (SDS) and denature proteins. all need diffCopyright. Cone © 2014 by Nelson Education Ltd. to form 38 micelles ↑ cme + can extract The protein  Predicting Membrane Spanning Segments DNA sequencing and protein prediction algorithms can be used to deduce the primary sequence. Transmembrane segments (TMs) are composed primarily of In an alpha helix, ~ 20 amino on hydrophobic amino acids. based a ation acids are needed to span the membrane. Strategy: scan the primary sequence for long stretches of hydrophobic amino acids representing TM segments. 39  drophobic nu Kyte-Doolittle Hydropathy Scale Residue Hydropathy index Residue Hydropathy index Isoleucine 4.5 Serine - 0.8 Valine Blanched 4.2 Tyrosine - 1.3 Leucine 3.8 Proline - 1.6 Phenylalanine 2.8 Histidine - 3.2 Cysteine/cystine 2.5 Glutamic acid - 3.5 Methionine 1.9 Glutamine - 3.5 Alanine 1.8 Aspartic acid - 3.5 Glycine - 0.4 Asparagine - 3.5 Threonine Tryptophan - 0.7 - 0.9 Lysine Arginine - 3.9 - 4.5 polat 40 &  Determining Protein Topology The hydropathy index for a stretch of amino acids can be determined by averaging the Kyte-Doolittle hydrophobicity values of all of the amino acids found in the segment. Move the window one residue to the right and recalculate the hydropathy index. Using the ProtScale online tool, you are able to generate the following hydropathy plot based on the primary sequence: hydrophon's Plot the hydropathy index vs. central amino acid in the sequence. treshold The “window size” can be changed: Smaller window à noisier plots polar Larger window à smoother plots know how to read How many transmembrane segments can be correctly identified using this plot? usually smaller than 202 odd number Answer: 7 the L14, slide 24. plots Look for peaks that are 20 amino acids long and very hydrophobic (high positive score). In this plot, there are 7 peaks between AAs 200-500 that meet this criteria, peaks before position 200 are stretches that are too short and only slightly 41 hydrophobic. 15. Ornithine is a metabolized derivative of the amino acid arginine. 10 mL of a 0.5 mg/mL solution of ornithine in water was mixed with 5 mL of octanol in a separatory funnel. At equilibrium, 1 mg was found in the octanol phase and 4 mg were found in the water phase. Calculate the log P value for ornithine: Answer: –0.3 L14, slide 15&16. = log [Conc.(octanol)/Conc.(water)] = log [(1mg/5mL)/(4mg/10mL)] = log 0.5 = - 0.3 Step 1. Assign hydrophobic value to each residue -1.6 -3.5 +4.5 -0.7 +3.8 +4.5 +4.5 +2.8 -0.4 +4.2 +1.9 +1.8 P E I T L I I F G V M A Step 2. Average values in 7 residue window; assign to central residue -1.6 -3.5 +4.5 -0.7 +3.8 +4.5 +4.5 +2.8 -0.4 +4.2 +1.9 +1.8 P E I T L I I F G V M A d Th arg = 1 6. fo The Step 3. Move one to the right and repeat… -1.6 -3.5 +4.5 -0.7 +3.8 +4.5 +4.5 +2.8 -0.4 +4.2 +1.9 +1.8 P E I T L I I F G V M A ↓ = 2 3 ang. 42  Identifying Transmembrane Segments Glycophorin A (GPA) Contains a single transmembrane segment between AA # 72-91. polar ac Bacteriorhodopsin Contains 7TMs, or positive peaks that span at least 20 amino acids. Copyright © 2014 by Nelson Education Ltd. 43  Generating Hydropathy Plots Several automated hydropathy programs available online for free: Technical University of Denmark - DeepTMHMM Tokyo University of Agriculture and Technology - SOSUI Adjustable window size: SIB ExPASy Bioinformatics Portal - Protscale SickKids - TM-Finder 45 BCH422H Key Messages Membranes are assemblies of amphipathic molecules held together by non- covalent interactions. Biological membranes are a heterogeneous mixture of lipids and proteins, with enzymes responsible for creating asymmetry within and across the lipid bilayer. Membranes are fluid-like asymmetrical structures; lipids can diffuse in the plane of the membrane and fluidity allows for proteins to carry out their various functions. Peripheral membrane proteins weakly associate with the membrane, while integral membrane proteins contain TMs and are more tightly bound. Salts, chaotropic agents and detergents can be used to isolate membrane proteins for purification. Integral membrane protein topology can be predicted using quantitative hydropathy scales. 46