PROTEINS AND CELL EVOLUTION CLASS 2B LEARNING OBJECTIVES To appreciate the structural and functional diversity of proteins Structurally proteins are very varied in size and shape. Functions of proteins include: Enzymes: catalyse covalent bond breakage and formation Structural: provide mechanical support Transport: carry small molecules or ions Motor: generate movement in cells and tissues Storage: store amino acids or ions Signal: carry extracellular signals from cell to cell Receptor: detect signal and transmit them to response machinery Gene regulator: bind to DNA to switch genes on or off To understand the structure of different proteins Amino acids are the subunits of proteins. A chain of amino acids is called a polypeptide. Each peptide has an amino terminal (N) and carboxyl terminal (C) which gives structural directionality. The side chains on amino acids create the different 20 amino acids. To be familiar with protein interactions and the concept of antibodies Denatured proteins may recover their natural shape (eg. After denatured due to urea, remove urea and protein refolds to original conformation). Chaperone proteins: guide the folding of new proteins to a correct conformation. helix: membrane bound proteins in secondary structure that cross the lipid bilayer in a spiral conformation. Complex structures: identical protein subunits that join to form structures such as dimers (2), helixes or rings. A single type of proteins subunit can form a filament (collagen and elastin), a hollow tube or a sphere. Disulfide bonds: very strong bonds critical in maintaining protein conformation. Antibodies: proteins that bind very tightly to their targets (antigens). Made of 2 identical light chains and 2 identical heavy chains. Production: B cells. Each B cell carries a different membrane bound antibody molecule on its surface, when antigens bind to this receptor the B cell is stimulated and secrete large amounts of the antibody. Function: help defend against infection. Antibodyantigen aggregates are ingested by phagocytic cells. Special proteins in blood kill antibodycoated bacteriaviruses. To appreciate how protein phosphorylation acts as a switch A conformational change in a protein can be driven by phosphorylation (P is removed from ATP and added to protein). In most cases phosphorylation turns proteins on. GTPbinding proteins are affected by a loss of a phosphate group creating a molecular switch. Changes in conformation allow a protein to walk along a cytoskeletal skeletal filament. Motor proteins use ATP hydrolysis to move in one direction along a cytoskeletal filament. To know the techniques by which proteins can be isolated and purified Proteins need to be purified to be studied. 1. This begins with rupturing the cell membrane creating a cell homogenate (extract). Sound: high frequency sound to break cells (ultrasound) Detergent: break apart lipids to make holes in the plasma membrane Force: use a needle and pressure to create a small hole in the plasma membrane Homogenizer: rotating probe plunger breaks cells via mechanical force 2. Proteins can be further purified and fractioned based on size or cellular localisation. Differential centrifugation: separates cell components based on size and density Velocity sedimentation: subcellular components sediment at different rates according to their size. Equilibrium sedimentation: cell components are separated based on their buoyant density which is independent of their size or shape. 3. Proteins can then be separated Column chromatography: permeable matrix of beads works as a solvent where proteins move faster dependent on their interaction with the matrix. Ionexchange: beads are charged causing proteins to bind Gelfiltration: beads are porous so larger molecules move through first Affinity: antibodies bind proteins Gel electrophoresis: electric field is applied to proteins, separating proteins on size. Isoelectric focusing: protein move to area so they reach isoelectric point (no net charge), separate based on charge. 2D polyacrylamidegel electrophoresis: separated based on isoelectric point and pH To understand the processes that are thought to have led to the evolution of selfreplicating cells Primeval sea (H 2) mixed with the primeval atmosphere (CH , H4, 2H an3 electrode) which caused amino acids to be detected. It is believed that RNA came first and directed protein synthesis. The evolution of new enzymes that synthesize nucleic acids created DNA and make RNA copies from it.