Class 1 Membrane Biological 1 _ 2024 (1).pdf

Biological Membrane Class I L.Spencer Topics: - Chemical Foundations - General characteristics - Structural characteristics of proteins - Isolation of Membrane Proteins. - Experimental Evidence (next class) 1- Chemical Basics: - Hydrophobic (non-polar molecules → aggregation) Insolubl...

Biological Membrane Class I L.Spencer Topics: - Chemical Foundations - General characteristics - Structural characteristics of proteins - Isolation of Membrane Proteins. - Experimental Evidence (next class) 1- Chemical Basics: - Hydrophobic (non-polar molecules → aggregation) Insoluble in water: Hydrophobic molecules. - Hydrophobic Links: The force that produces the bond between non-polar molecules. These molecules are not able to form bonds with water. - Non-polar molecules can be bonded to each other, although in weak form through Van der Waals type interactions - Polar molecules: Dissolve in polar solvents as water and non-polar solvents such as Hexane. 2- Phospholipids are amphipathic molecules: Multiple non-covalent bonds are also essential for stabilizing the structure of bio-membranes. Main components: Phospholipids. Phospholipids: One or more chains of fatty acids. (Chain of hydrocarbons bound to a carboxyl group (-COOH). amphipathic molecule in aqueous solution Micelle Formation Fatty acids: Saturated (without double bond) Unsaturated (at least one double bond). Please, Who can explain this figure? This table present the most common lipids into the membrane The Cis double bond introduces a straight rigid angulation in the hydrocarbon chain Formation of phospholipids: 2 chains of fatty acid groups bound to very small hydrophilic groups (polar head). Phosphate Amphipathic molecule: Why is a phospholipd amphipathic molecule? TO ISOLATE THE ´PROTEIN FROM BIOLOGICAL MEMBRANE Form organized structure into the water micelle Sheet of the bilayer Liposome Triacylglycerol Glycerol Phospholipids Where yellow are fatty acid groups Eg.: Phosphatidylcholine  Class of phospholipids : (Figura 5-27) Phosphoglycerols Ethanolamine and Serine (Attached to alcohols). Inositol (Linked to sugars). Fatty acid side chains are esterified by 2 or 3 hydroxyl groups of glycerol. The latter group is esterified by a hydroxyl group of another hydrophilic compound glycolipid Phosphoglycerol Sphingolipid Two faces of the a cellular membrane as the Cytosolic face and the exoplasmic face  Steroids: (cholesterol and its derivatives) → anphipatic Basic structure is a hydrocarbon and 4 rings (abundant in eukaryotic membrane) General structure of a steroid: Cholesterol Hydrophilic region Hydrophobic region Curved shape High concentration of sphingolipid Increase its stability Table of lipid composition: Each type of membrane has characteristics of lipids and proteins. The relative proportion of them depends on the membrane. Other components of membranes Glucoproteins. Fig 3-32 (3) Glucolipids or Glycolipids. Carbohydrates attached to sugars increase the hydrophilic character of lipids and proteins and contribute to stabilize the conformations of many membrane proteins. Classification of proteins Surface or peripheral. Integral or Intrinsic. On the outside Function intervene in signals Protein domains Intracellular F. (pores or channels) Protein domains F. anchorage of the protein of the cytoskeleton in the cytosolic face F. Intracellular signaling. Schematic diagram of the typical membrane proteins of the biological membrane Plasma membrane defines the cell and separates the inside from the outside.  Schematic diagram of the membrane proteins typical of the biological membrane the phospholip bilayer serves as a permeability barrier Types of membrane proteins 2 Protein classes Peripheral of Membrane Integrals (Transmembrane proteins) Transmembrane or integral proteins: - Domains that cross the membrane (lipid bilayer) - Domains that are : α helix Multi stranded β outside inside What type the a.a are? Classification of amino acids Peripheral Proteins: - They do not interact with the hydrophobic core or nucleus. - They bind to the membrane indirectly through interactions. I. Integral Interactions II. Interactions with the polar heads of the lipids from bilayer E.g. Spectrin (Cytosolic Face of GR) and Actin Cytoskeleton Eg. Protein-kinase C (Intervenes in the translation of signals) Eg. Protein from the Extracellular Matrix (Protein Integrins) Erythrocyte membrane integral proteins: Domains α helices Hydrophobic interaction with helices of the bilayer Ionic interactions with the Polar groups of the bilayer Eg. Glycophorin in RBC (Plasmodium merozoite invasion) Fig. 3-33 Both types of interactions Hydrophobic ( Van der Walls) Ionics (a.a. Lysine and Arg +), Avoid the Protein’s Displacement Identific the parts of the biological membrane Example: Glycophorin A  Fig. 3-34 Structure of Bacterodopsin, contains 2 or more alpha-helices that cross the membrane. - Prot. Multiple strands of Porins They form barrels (channels) that pass through the membrane. Porins: transmembrane proteins They provide channels for the passage of disaccharides and phosphates in E.coli. By X-Ray Porins are trimers RECEPTORS in the plasma membrane are proteins that allow the cell to recognize chemical signals present in its enviroment  E.g. Glicocyl Phosfatidil Inositol (Fig. 3-36) Eg. Glicosyl phosfatidil inositol Plasmodium invasion + MSP-1 (Parasite surface protein one) What is this figure? Purification and characterization of membrane proteins Purification and characterization of membrane proteins Proteins vary in: -Charge - Size - Water solubility That is why you have to use several methods of isolation Extraction and Purification Techniques - Detergent Action Types of detergents Ionic (Charged Groups): (SDS) dodecilsulfate. Non Ionic (no charge): Triton X-100. Used Micelle Formation Each detergent has its minimum micellar concentration(CMM) SDS Electrophoresis or SDS -PAGE Calculation of the relative weight of a protein by SDS-PAGE analysis (polyacrylamine gel electrophoresis) It is calculated using the Rf of a reference protein and the rf of the problem (sample) protein. The Rf is defined as the migration distance of the protein through the gel divided by the migration distance of the dye front. Three liquid chromatography to separate proteins based on their mass, charge or affinity to a ligand  Result Western Blotting We will made in the Lab ELISA TEST Immunofluorescence to locate protein in the cell Microscopia confocal https://www.youtube.com/watch?v=moPJkCbKjBs&ab_channel=JCCCvideo  Next Class - Fluid Mosaic Pattern - Experimental Evidence - Discussion of membrane revision article (virtual classroom) Answer the following questions based on the Singer and Nicolson model article: 1- How do membranes exist, in which states? 2- What is the purpose of this review? 3- How did Singer and Nicolson see the model? 4- Say an evidence for the bilayer? 5- How can membrane proteins be associated? 6- In the model, which molecules can be in constant motion? 7- What types of movements occur in this model? 8- What do you mean by FLUIDITY? 9- What is an amphitropic protein?