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MCDB 310 1st Edition Lecture 5 Outline of Last Lecture I. Protein biochemistryII. Protein purificationa. Purification by chargeb. Purification by sizec. Purification by affinity for a ligandd. Electrophoresise. Protein analysis (how do we know that the protein we have collected is pure?)III. 4 Levels of protein structurea. Primary structure Outline of Current Lecture I. 4 levels of protein structure (continued from last lecture)a. Secondary Structure: alpha helices and beta sheetsb. Tertiary Structurec. Quarternary Structured. Analyzing protein structure in relation to its functionII. Two major types of protein (fibrous and globular)III. Protein stability and foldingIV. Function of globular proteinsCurrent LectureThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.I. 4 Levels of protein structure (continued from last lecture)a. Secondary Structurei. From Ramachandran Plot, there are only a few possible secondary structures possible due to favorable phi/psi angle combinations1. Each amino acid can have its own Ramachandran plot because it is dependent on the eclipsed/staggered conformations of the R groupa. Lysine has the most possible phi/psi angle combinationsb. Proline has the least possible phi/psi angle combinationsii. Alpha Helices (1st example of possible secondary structure):1. Helical backbone is held together by hydrogen bonds between the backbone amides of nth and n+4th amino acids2. There are about 3.6 residues per turn of the helix3. Always right handed (follow the right hand rule)4. Peptide bonds are parallel to the helical axis (what the helix is wrapping around)5. R groups therefore are roughly perpendicular to the helical axis (see diagram below for top view of alpha helix)a. Because 3.6 times 2 is approximately 7 —> under amino acid #1 will be amino acid #8 (n+7 is directly under nth amino acid)b. This is important because these amino acids directly on top of each other can interact ionically (if they are charged) and form a salt bridge, or hydrophobically (if both are hydrophobic) to form a Coiled Coil (several alpha helices interacting with one another)6. The Helix dipole:a. Because the peptide bond has a strong dipole across it, all the individual dipoles add up to form a large dipole through the whole helix (partial negative charge at carboxyl terminusand partial positive charge at the amino terminus)b. To stabilize the charges at the end, helpful to put a negatively or positively charged amino acid at either endiii.Beta Sheets (second example of possible secondary structure)1. Pleated sheets form because of the planarity of the peptide bond and tetrahedral geometry of the alpha carbons2. Hydrogen bonds between the backbone amides in different strands come together to form the pleated sheeta. Antiparallel beta sheets form when strands run opposite in directions (STRONGER hydrogen bonds because they are perfectly linear)b. Parallel beta sheets form when the strands run in the same directions (WEAKER hydrogen bonds because the hydrogen bonds are not linear but bent)3. R groups fall either above or below the sheetiv.How can we determine secondary structure? (is it alpha helical, beta sheet, or a mixture of both?)1. Use Circular Dichroism (CD): a form of spectroscopy that looks at how proteins react to circularly polarizedlighta. Alpha helices and beta sheets have very different spectrab. Combination of the two secondary structures show up as roughly an average of the two spectra2. Most proteins are a mixture of both types of secondary structureb. Tertiary Structure: overall spatial arrangement of atoms in a proteini. Structure is formed when all the secondary structures cometogether and form an interactionii. Interacting amino acids are not necessarily next to each other in the amino acid sequencec. Quarternary Structure: formed by the assembly of individual polypeptides into a large, functional clusteri. Again, the non-covalent and covalent (disulfide bonds) interactions are what form a quarternary structureii. Not all proteins have a quarternary structure iii.Individual polypeptides are called subunits or protomersiv. Whole polypeptide in quarternary structure is called an Oligomeric/Multimeric polypeptided. If we know the 3D structure of a protein, we can understand how it performs its functioni. 3D structure: the x,y,z coordinates of every atom ii. Protein Structure Methods: 1. X Ray Crystallography:a. Must purify the proteinb. Crystallize the protein (still don’t know fully howit works)c. Collect X Ray diffraction datad. Solve the crystal structuree. With this method:i. There are no size limitsii. Very well established methodiii.But one cannot see hydrogen atomsiv.Also difficult to get the crystal structure (usually only a 10% chance that a crystal will form)2. Biomolecular NMR:a. Purify the proteinb. Dissolve the protein in a bufferc. Collect NMR data for H (proton), C 13, and N 15nuclear spins (incorporate these isotopes into the protein)d. Assign NMR signals to specific atoms/bondse. Solve the protein structuref. With this method:i. There is no need to crystallize the proteinii. One can see many hydrogensiii.But there is a size limit (this works best with smaller proteins-anything below 80 kilodaltons (800 amino acids long))II. Two major classes of proteins (based on tertiary structure)a. Fibrous Proteins: analogous to a rope (strong, rigid, textured)i. Keratin1. Simplest possible structure: just one long alpha helix2. That simple helix then forms a two-chain coiled coil (due to hydrophobic interactions)3. Come together to form a protofilament which come together to form a protofibril4. Helices are held closely together by disulfide bond (due to the presence of Cysteines)a. Through a reduction reaction, the disulfide bonds will breakb. The filaments will wiggle alongside each other (curl)c. Add an oxidizing agent, and thedisulfide bonds will reform(permanently curling the hair)ii. Collagen1. Each chain is a long Gly and Pro rich LEFTHANDED HELIX (very unique)2. Three collagen chains intertwine into aright handed superhelical triple helix (See diagram atright)3. Packing these superhelices together forms a fibril4. 4-Hydroxyproline in collagen:a. Post-translational processing adds a hydroxyl group to the prolineb. This causes the conformation to change (forms a favorable pucker)c. Able to form a lot more hydrogen bonds that stabilize this protein


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