Biological Membranes: Chapter 11 Biological Membranes: Part 1

Part 1 and 2 Learning Goals: To Know Membranes Part 1 and 2 Learning Goals: To Know The function of biological membranes The structure and composition membranes and their molecules Dynamics of membranes Structure and function of membrane proteins Transport across biological membranes

Why are TEMs of membranes always light in the middle and dark on the sides? 50 – 80 Å Thick Membranes have this double track appearance in TEM. The white-clear inner layer does not stain with the heavy metals used in TEM staining. Why do these stains stain the edges but not the inner part? EOC Problem 5 has to do with the length of a fatty acid: how does this play into the thickness of this membrane?

Cells Are Loaded with Membranes Pancreatic cell: loaded with secretory granules, but some space for mitochnordia, er and nucleus. What’s the answer to the previous thought question?

Major Membrane Lipids Note that biological membranes are loaded with protein…but it is the phospholipid that is the fluid matrix of the membrane. Note the phylogeny of sterol types in the membrane.

Membrane Lipid Composition – Rat Hepatocyte Note that the lower three have almost identical lipid composition. Why? The plasma membrane is loaded with cholesterol. Why?

Fluid Mosaic Model The standard fluid moscaic model…it looks way to orderly (see some other cartoons later). Proteins are either transmembrane or peripheral (on either side). The phospholipids are in two layers or “leaflets”.

Lipids aggregate into structures in water Structures formed depend on: type of lipid concentration Micelles Liposomes Bilayers

Lipids in Water Lipids in water tend to form structures that “hide” the non polar parts from water. These are artificial membranes formed in the lab by simply taking some purified phospholipids (which have a wax-goo consistency) and then passing ultrasound through the liquid to force water-lipid interaction. Fatty acids form micelles, phospholipids form membranes or liposomes. EOC Problem 3 … Number of SDS moleucles/micelle. You will need to calculate the MW of dodecyl-sulfate.

Asymmetric Distribution of P-lipids in Erythrocyte Membrane This is important. See that phosphatidylethanolamine and phosphatidylcholine are very asymmetrical in opposite ways. And the last 4, though small in amount, are almost in the inner leaflet.

Membrane composition and asymmetry FIGURE 11–6 The distribution of lipids in the membranes of a typical cell. Each membrane has its own characteristic composition, and the two monolayers of a given membrane may differ in composition as well.

Membrane Asymmetry Derived in ER These are delightfully named enzymes: flippase, floppase and scramblase (any analogy to this course?).

Functions of Proteins in Membranes Receptors: detecting signals from outside Light (opsin) Hormones (insulin receptor) Neurotransmitters (acetylcholine receptor) Pheromones (taste and smell receptors) Channels, gates, pumps Nutrients (maltoporin) Ions (K-channel) Neurotransmitters (serotonin reuptake protein) Enzymes Lipid biosynthesis (some acyltransferases) ATP synthesis (F0F1 ATPase/ATP synthase)

Extraction of Membrane Proteins Peripheral proteins are more easily purified that transmembrane proteins. The transmembrane proteins when purified are always contaminated with the detergent used to disrupt the membrane or phospholipid.

Glycophorin – Transmembrane Protein Tetra-Saccharides, N-linked Tetra-Saccharides, O-linked Glycophorin is a major erythrocyte protein (there are about one million glycophorin molecules in one erythrocyte) that often is found in dimers (shown here is a monomer). It is highly decorated with oligosaccharides, bot O-linked and N-linked. Check out the part in the membrane: all alpha helix non-polar amino acids, the part with polar R groups are in the non-alpha helix bend by the outside and the two E’s are neutralized by the two H’s, and the S alcohol is quite happy in a non-polar environment. Check out the amino acids immediately following on the inside (96 through 105) and outside (55-61) polar environments: the aa’s are all very polar. These two features keep the protein “dissolved” in the membrane and water on either side.

Six Types of Integral Membrane Proteins These types are not necessary to know, but demonstrate all the sorts of possibilities of a protein being transmembrane. Type III is like a snake going in and out…and are some times called serpentine proteins.

Bacteriorhodopsin Bacteriorhodopsin we will see later as a proton pumping protein. This has very little of the protein in contact with water compared to Bacteriochlorophyll, next slide

Bacteriochlorophyll – Reaction Center Chlorophylls in Red Here is a transmembrane protein loaded with chlorophyll molecules…and surrounding them in the reaction center. Although many chlorophyll molecules are here, only one starts the light induced electron flow. The others are light antennae to pass the energy to the reaction center chlorophyll.

Great Illustration of Phospholipid as a Fluid Sec A (chaperone) in blue ribbon. Cytoplasmic side with ATP (bright light) Sec YE in Cream Space-filling cut away to show channel Transported protein in red thread This is a much more realistic cartoon of the phospholipid: you can see their fluid nature. The cytoplasm is on top and SecYE is a protein translocating transmembrane protein. Note that when a protein (orange) is going across a membrane, it is chaperoned (blue ribbon structure) to keep it from folding so it can then be transported as a single strand through the transporter (this takes energy) then begins to fold on the outside (bottom of the page).

Another Great Illustration of Phospholipid as a Fluid G Protein – major signaling protein, next Chapter. Here the cytoplasm is below the membrane. We will work with this protein in Chapter 12.

Phospholipid Associated with Isolated Membrane Proteins Purified transmembrane proteins come with detergent or phospholipids. This has made their crystalization and structure determination lag behinds those of regular water soluble proteins. Sheep Aquaporin Fo Portion of V type Na-ATPase

Hydropathy Plots Back to Chapter 3: each amino acid has a hydropathic index (Table 3-1) the positive numbers are hydrophobic and the negative numbers are hydrophilic. See next slide.

Remember Hydropathic Index? Table 3-1. Non Polar R groups have Positive Hydropathic Indices Y and W are negative

Polar Amino Acids have Negative Hydropathic Indices

Hydropathy Index Calculated from a Moving -Window of 7 to 20 Amino Acids Moving window gets the average of the window amino acid and plots the number at the position of the middle amino acid. Thus, it gives an index of the amino acid and its vicinal aa’s. So, it is easy to see that glyophorin has a membrane spanning region (has to be hydrophobic over a long enough stretch to span a membrane. Go back to slide 16 to see the 7 membrane spanning regions of Bacteriochlorophyll.

Transmembrane Proteins – Positions of Y and W. Polar R – groups are Blue W and Y have a unique position in many membrane spanning proteins. A factoid. Y is Orange W is Red

Membrane Spanning Regions can also be Beta-Barrel Structure This is to show that membran spannig regions are not always alpha helix. Beta barrels are usually specific pore involved in transport.

Another Great Illustration showing the Membrane as a Fluid Surface of a B-cell showing the B-cell Receptor (purple, mono-valent IgM) binding to an HIV particle.

Sided Proteins Anchored by Fatty Acids (Inside) Some peripheral proteins are attached with fatty acids or isoprenes. The GPI anchor uses sugars to link to a phosphatidylinositol.

Membranes can transition from Gels to Fluids a. Gel State b. Liquid Ordered Liquid Disordered Membranes are only functional in the Liquid Ordered state.

We can’t do this, but bacteria have to live at the temperatures in which they find themselves. This experiment grew the bacteria at the temperatures indicated then measured the fatty acid composition of the membrane. Conclusion: bacteria can adjust the fatty acid content of the membrane to make sure it is in a fluid ordered state at a wide range of temperatures. But, what would happen if you took cells grown at 40oC and rapidly switched the temperature to 10oC; and doing this in the reverse, taking 10oC grown cells and immediately switching the temperature to 40oC….what would happen.

Measuring Rate of Phospholipid Mobility This was the first experiment that showed fluidity: by measuring the rate of red dots entering the bleached area (containing phospholipids that had non-fluorescent-oxidized dyes, they were measuring the actual motion of phospholipids in, out, around each other.

Membrane Lipid Movement over 56 msec This can now be done with tracking confocal microscopes. It shows the motion over 56 msec: one phospholipid bounced around in one area, then another, then another over 0.056 sec. AND, this is “ordered” fluidity !! EOC Problem 6 involves temperature effects on lateral diffusion in membranes.

Some Membrane Proteins Have Cytoskeleton Restricted Movement This is important…the phospholipids are highly mobile, but the proteins are not as mobile. What keeps them in place is links to other structural proteins: cytoskeleton on the inside and proteoglycans and other proteins on the outside. Membrane proteins have to be in the right place in the cell to function (we will see this in the next part of this chapter with the example of glucose transporters in the small intestine epithelial cells).

Atomic Force Microscopy Atomic force microscopy shows surface features of membranes.

AFM Aquaporin from E. coli – Outside View AFM of an aquaporin over expressed in Escherichia coli with the inset being amplifed. This is a special case, a more normal eukaryotic plasma membrane is next.

Rafts and GPI-linked Proteins Rafts have a greater content of cholesterol and sphingolipids. The proteins really are not “spikes”….a feature that is an artifact from the cantilever being tossed up above a protein and then rapidly back down as it scans across the membrane. The model of this is next slide. Rafts and Sea

AFM Detects “Rafts” of Sphingolipids + Cholesterol The sphingolipids are green. See that the GPI linked protein is really globular rather than a “spike”.

Hierarchy of raft-based heterogeneity in cell membranes Hierarchy of raft-based heterogeneity in cell membranes. (A) Fluctuating nanoscale assemblies of sterol- and sphingolipid-related biases in lateral composition. This sphingolipid/sterol assemblage potential can be accessed and/or modulated by GPI-anchored proteins, certain TM proteins, acylated cytosolic effectors, and cortical actin. Gray proteins do not possess the chemical or physical specificity to associate with this membrane connectivity and are considered non-raft. GPL, glycerophospholipid; SM, sphingomyelin. (B) Nanoscale heterogeneity is functionalized to larger levels by lipid- and/or protein-mediated activation events (e.g., multivalent ligand binding, synapse formation, protein oligomerization) that trigger the coalescence of membrane order–forming lipids with their accompanying selective chemical and physical specificities for protein. This level of lateral sorting can also be buttressed by cortical actin. (C) The membrane basis for heterogeneity as revealed by the activation of raft phase coalescence at equilibrium in plasma-membrane spheres. Separated from the influence of cortical actin and in the absence of membrane traffic, multivalent clustering of raft lipids can amplify the functional level to a microscopic membrane phase. Membrane constituents are laterally sorted according to preferences for membrane order and chemical interactions. D Lingwood, K Simons Science 2010;327:46-50 Published by AAAS

Integral Membrane Proteins Involved in Cell-Cell Communication Many types of cell surface protiens can specifically attach to other cells or proteoglycan connective material.

Membrane Fusion Events, An Overview To do this, proteins have to get two membranes very close together so they touch.

Caveolin Dimers form Caveloa Caveloin is one type of membrane shaping protein that causes the membrane to begin invagination.

Three Models for Protein-Induced Curvature Protein super-structure is of essence here. There are many proteins that are actually guiding this process, next slide.

Types of Endocytosis Several of these RAB proteins have been discovered by FIU’s own Dr. Barbieri whose lab works on the secrets of how these proteins can bring about the different types of endocytosis. The important thing here is that caveloin and clatherin are major players in forming the invagination, there is another set of proteins involved with getting the vessicle pinched off from the plasma membrane and then another set, the RABs to get them to fuse in they cytoplasm to form the early endosome.

Influenzia Virus Must “Uncoat” in Cytoplasm Influenza virus is one of the enveloped (covered by a membrane) viruses. The viral transmembrane proteins recognize the host cell and bind to its receptor. Then after endocytosis, the cause the viral membrane to fuse with the host membrane thereby delivering the viral RNA into the cytoplasm…the job of the H-protein (or HA in this diagram).

Things to Know and Do Before Class The major and minor membrane phospholipids. Transmembrane protein structures and how they are held into the membrane. Peripheral membrane proteins and how they are attached to the membrane. Membrane temperature driven transitions. What AFM tells us about membrane structure. Membrane fusions + formation of endosomes. EOC Problems: 3, 5, 6 (Hint need to calc SDS mole wt 288 Daltons)