Biochemistry 3 Nonenzymatic Protein Function and Protein Analysis

Biochemistry 3: Nonenzymatic Protein Function and Protein Analysi Cellular Functions Protein Isolation Structural Proteins: Compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix. The most common structural proteins are collagen, elastin, keratin, actin, and tubulin. They are generally fibrous in nature. Motor Proteins: Have one or more heads capable of force generation through a conformational change. They have catalytic activity, acting as ATPases to power mvmt. Common applications include muscle contraction, vesicle mvmt within cells, and cell motility. Examples include: myosin, kinesin, and dynein. Binding Proteins: Bind a specific substrate, either to sequester it in the body or hold its concentration at steady state. Cell Adhesion Allow cells to bind to other cells or surfaces. Molecules (CAM): Cadherins: Calcium dependent glycoproteins that hold similar cells together. Integrins: Have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix. Selectins: Allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system. * See appendix for detailed information Electrophoresis: Uses a gel matrix to observe the migration of proteins in responses to an electric field . Native PAGE: Maintains the protein’s shape, but results are difficult to compare because the mass / charge ratio differs for each protein. SDS-PAGE:Denatures the proteins and masks the native charge so that comparison of size is more accurate, but functional protein cannot be recaptured from the gel. Isoelectric Separates proteins by their isoelectric point (pI); the Focusing: protein migrates toward an electrode until it reaches a region of the gel where pH = pI of the protein. Chromatography: Separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase. Column Uses beads of a polar compound (stationary phase) with Chromatography: a nonpolar solvent (mobile phase). Ion-Exchange Uses a charged column and a variably saline eluent. Chromatography: Size-Exclusion Relies on porous beads. Larger molecules elute first Chromatography: because they are not trapped in the small pores. Affinity Uses a bound receptor or ligand and an eluent with free Chromatography: ligand or a receptor for the protein of interest. Antibodies: Immunoglobulins, Ig. Used by the immune system to target a specific antigen, which may be a protein on the surface of a pathogen or a toxin. The variable region is responsible for antigen binding. Biosignaling Protein Analysis Ion Channels: Can be used for regulating ion flow into or out of a cell. Ungated Channels: Always open. Voltage-Gated Channels: Open within a range of membrane potentials. Ligand-Gated Channels: Open in the presence of a specific binding substance, usually a hormone or neurotransmitter. Enzyme-Linked Participate in cell signaling through extracellular ligand Receptors: binding and initiation of 2nd messenger cascades. G Protein-Coupled GPCR has a membrane-bound protein called the GReceptors: Protein (a, b, g subunits). The 1st messenger ligand initiates the 2nd messenger and the cascade response. G-Protein Coupled Receptor Structure: Primarily determined through x-ray crystallography after the protein is isolated, although NMR can also be used. Amino AcidDetermined using the Edman Degradation. Sequence: Concentration: Determined colorimetrically, either by UV spectroscopy or through a color change reaction. Bradford Assay, BCA Assay, and Lowry Reagent Assay each test for protein and have different advantages and disadvantages. The Bradford Protein Assay is most common. It uses a color change from brown-green blue. Beer-Lambert Law: Absorbance=+ e+𝐶+𝑙 e+=extinction+coefficient 𝑙 =path+length+in+cm Notes: Epinephrine is a ligand 1st messenger. At the end of the GPCR process, Phosphodiesterase deactivates cAMP and GTP hydrolyzed back to GDP. 𝐶 =concentration