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SC BIOL 541 - Types of Helices. Protein Purification.
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BIOL 541 1st Edition Lecture 3 Outline of Current Lecture Secondary and tertiary structures. Protein purification.Current LectureSecondary structures:3. Helix structure:a. Alpha helix: This is a common helix with greatest hydrogen bonds for width and stability. Nitrogen and oxygen of the carbonyl of turn has hydrogen bonds, also carbonyl is on the same side and causes dipoles. Just like many small magnets join to form a large magnet, so do many dipoles form a large alpha helix. There is coil distortion due to 7th residue superimposed on one another.b. Poly proline helix: This is the only stable helix for proline. Collagen helix is the common type. This L- handed helix is tighter – 3 strands twist across and triple helix. There are small spaces with monotonous triplet sequence repeat of Gly- Pro- Hyp.c. Hydroxy proline: It has less hydrogen bonding and therefore is an unstable helix. The triplet helix gives it stability with protein- protein association and hydrophobic interaction.d. pi helix: It is a short, fat, and squat helix. It has an extra amino acid than alpha helix. It has successive turns that are condensed. There are enhanced dipole and ligand binding. (-) charged amino acids clustered together to make excellent metal binding sites or ligand sites.e. 3/10 helix: It has exactly 3.0 amino acids/turn. It is the smallest helix not found in mature protein. It is intermediate during protein folding. It can establish helix quickly due to its small structure. It can be remodeled into alpha helix.Tertiary structures:1. Fibrous protein: These are elongated proteins-collagen with intermediate filaments-keratin forming 1 long alpha helix.2. Globular protein: It is a silk protein with beta- pleated sheet, whereby multiple domains fold on themselves to form globular protein.Alpha helical bundle: It can be parallel or anti- parallel. Example, hemoglobin has a spherical structure where there are twists and turn.Beta strands: These are sheets where beta strands go up and wrap with alpha helix andcome around to form cylinders. Example, the sandwiches lie flat with sheets twisted and roll up to form “beta- barrel”. In other words, beta- band in center with alpha helix circling them.Motif (fold): This is a folding pattern involving 2 or more elements of secondary structures and connection between them.Single peptide chain- one level only.Multiple peptide chain: This is a quaternary structure comprising of 4 homologous units- 2 alpha and 2 beta. There are alpha beta pairs in binding with non covalent bonds. A biological molecule is the hemoglobin.Fibrinogen: This is a precursor molecule in blood clot formation where fibrinogen converts to fibrin. Fibrinogen has multiple subunits such as alpha 2, beta 2, and gamma 2: Where alpha beta as the amino terminal and tryptophan as the extension.One alpha, beta and gamma as intact structure and another alpha, beta and gamma as another structure: Together they form v-shaped structure with disulphide covalent bonds. Domain: This is part of the polypeptide chain that is independently stable and undergoes movements.Fibrinogen: 4 parts:Domain 1:The amino terminal connects 2 halves. The high proline content disrupts thesecondary structures. Once heavily sulphated (-) charged random structure interfere with packaging to form strands (blood clot forming).Domain 2: Alpha, beta and gamma each for alpha helix. The proline content decreases as it is helix breaker. Helix twist around each other as fibrous protein. At the end of domain 2, beta and gamma come to normal level of proline, thus breaking into individual structures that are globular proteins.The carboxyl terminal of alpha subunit has high proline and hydrophilic residue that forms the free swimming tail.Lysine and glutamine juxtapose to form isopeptide bonds and firm up the strand.Next strands in all directions form networks.Carboxyl free terminus tail can reach up to form covalent bond with RBC.Protein Purification: This is based upon solubility property of proteins whereby polarity aspect is established for protein derivation. Compounds are added that have a high affinity for water, hence outcompete for water. Since there is water sphere around the compound and not the protein, the protein comes out. Solubility factor of salt compounds and charge density factor is used for protein extraction.1. Ammonium sulphate: This is added with rapid stirring to enable protein derivation. The compound is added gradually in concentrations till 10% saturation. The solution becomes turbid and precipitate forms. In the supernatant add the compound till 10 –20% level is reached in order to equilibrate. Assess fractions to see protein – if protein is observed at 40-50% level, the second time, go directly to 30% to judge protein calibration.Fibrinogen---hemoglobin-myoglobin (most soluble) For biological protein molecules, no resolution for purity is seen. Only at high concentrations, precipitate is enacted. However, the precipitate formed using ammonium sulphate compound is stable, hence can be used for long term storage.2. Using organic solvents to displace water: Water miscible organic solvents such as ethanol, methanol, andacetone can be used.a. Acetone: 10% solution gives turbidity. In the supernatant 20% acetone is added.Disadvantage:Since protein is sensitive, denaturation can occur. As the hydrophobic structures are away from water, denaturation destroys the folding.Advantage: It is generally used to delipidate the sample. It gives an aqueous extract of protein. In chromatography, the columns are smeared by lipids, therefore lipids need to be extracted.b. Polyethylene glycol: The solubility index is between the salt compound and organic solvent and hence at 10% turbid solution is seen. Supernatant needs 10% to assay the protein. There are charges and polarity aspects that interefere with the protein, hence charges are removed. In general, (+) and (-) charges are observed, whereby excess (-)


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