Biochemistry BBMB 301 Week 2 Notes
Biochemistry BBMB 301 Week 2 Notes BBMB 301
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This 6 page Class Notes was uploaded by Emily on Monday February 8, 2016. The Class Notes belongs to BBMB 301 at Iowa State University taught by Robert Thornburg in Spring 2016. Since its upload, it has received 22 views. For similar materials see Survey of Biochemistry in General Science at Iowa State University.
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Date Created: 02/08/16
Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 4 – CHAPTER 4 – PROTEIN STRUCTURE By: Emily Settle Protein Structure Visualization - http://www.umass.edu/molvis/tutorials/hemoglobin/index.htm Formation of peptide bonds o Nucleophilic substitution of carboxyl group oxygen with amino group nitrogen Dehydration of carbonyl/amino moieties Produces amide group Produces dipeptide Building blocks linked into polymers. Polypeptides: multiple peptide bonds link amino acids into polymers o Unique orientation Free amino group at one end – N terminus Free carboxyl group at the other – C terminus o Primary structure - sequence from N to C terminus Disulfide bonds – formed by two cysteine residues o Oxidation/reduction reaction (RedOx) o Distinct from most interactions in proteins Covalent – most interactions are weak bonds like ionic, hydrogen, hydrophobic, van der Waals. Between chains or links within chains Intrachain – within one chain Interchain – between two chains Ex insulin has both Resonance of Amide Groups o Electrons delocalize between N, C, and O of the amide group Partial double bond character C-N bond length shorter than a single bond Six atoms in a single plane No rotation between the C and N of amide group Amide Bonds in Trans Configuration o Partial double bond reduces steric interation between R groups Some exceptions Proline sometimes exists in both cis and trans configuration Proline cis-trans switch is sometimes a control mechanism Conformation: Rotation around single bonds o Bonds to Cα in each amino acid can rotate – amide bond planes rotate relative to each other. o Large number of conformations possible for a protein. o Restrictions on bond rotation: All rotation angles possible but… only a subset of combinations actually occur. Combination – N-Cα rotation angle (Φ) paired with Cα-CO rotation angle (Ψ) Steric hinderance prevents the other combinations from occurring Secondary structure – structural untis created by repeating patterns of rotation angles, stabilized by H bonds between atoms of the main chain. o H bonding atoms are O and HN- of the amide group o R groups extend away from the secondary structural element o Discrete sections of the polypeptide form secondary structure units Certain amino acids delineate start and stop of the element Loops and turns connect the secondary structural element o Types – α helix, β pleated sheet α Helix: some polypeptides can fold entirely into α helices connected by loops. R groups perpendicular to the helix axis. β Pleated Sheet: Structure can be visualized as a flat sheet (not truly flat) R groups extend from the sheet on top and bottom. Wraps and twist to form a β barrel structure Turns: four amino acids (often Pro and Gly), reverse direction of chain in space Loops: large and variable Amide atom H bonding Tertiary Structure – tertiary structure is the overall 3D structure of a polypeptide o created by interactions between R groups often between atoms far apart in the primary structure folding into tertiary structure brings distant amino acids closer together so they can work together (active site) o hydrophobic faces of helices and sheets brought together in interior core o polar residues often on surface o active sites in crevices or pockets o discrete functional segments – domains folds seen repeatedly in many proteins o Hydrophobic (water-hating) residues aggregate in interior core o Hydrophilic (water –loving) residues on surface (often) o Active site residues in crevices or pockets (often) Myoglobin o Prosthetic Group – heme found in Myoglobin o Enhances chemistry that the protein can accomplish o Heme binds O2 heme protein can become an O2 binding protein. How do hydrophobic residues effect protein structure o Polar residues on surface of trypsin o Allows interaction with water o Hydrophobic residues in interior o Oil and water don’t mix. Stabilize Tertiary Structure o From R group atoms Hydrophobic interactions Electrostatic bonds Salt bridges or water bridges Asp, Glu, Lys, Arg H bonds Covalent bonds Disulfide bonds between Cys residues Quaternary Structure – interactions between individual polypeptides o Protein – functional unit o Polypeptide – chain of amino acids o Polypeptide = protein can also include a prosthetic group o Protein = multiple polypeptides = quaternary structure with or without prosthetic groups o Same stabilizations as tertiary structure listed above. o Structure ex Hemoglobin 4 polypeptides 2 of one kind and 2 of another (α2β2) 4 prosthetic groups: one heme in each polypeptide o Denaturation Loss of secondary and tertiary structure from breaking weak bonds and disulfides. Primary structure retained! Agents and WHY Acid/base – disrupts H bonds H bonding compounds such as urea (caotropic agent) – disrupts structure of water, H bonds. Detergent, organic solvents – disrupts hydrophobic interactions Reducing agents – breaks disulfide bonds Salt Heat – general disruption o Renaturation Ribonuclease regains function if denaturing agents are removed slowly (dialysis) Regaining function indicates that native structure has also been regained during renaturation. This indicates primary structure of a polypeptide determines the secondary and tertiary structure. Sometimes helper proteins are needed to allow a denatured polypeptide to fold into its native structure o Chaperone protein Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 5 – CHAPTER 5 – TECHNIQUES OF PROTEIN PURIFICATION By: Emily Settle Genome – nucleotide sequence of all genetic material in an organism o Human genome – 3,000,000,000 base pairs Gene – region of DNA that specifies a functional product (usually protein, sometimes RNA) o Human genome – 19,600 genes Proteome – subset of proteins present in any given cell or tissue o 5,000 different proteins in proteome with overlapping subsets Protein purification – proteins studied as individual entities o Must be purified to homogeneity o Steps in purification Fractionation of subcellular compartments Differential precipitation owing to salt concentration Chromatographic separations Based on size Based on charge Based on affinity to immobilized ligands o Degree of purification – amount of target protein normalized to the amount of total protein. Detect presence of the desired protein o Activity assay Many proteins are enzymes that catalyze chemical reactions Reaction products can be detected spectrophotometrically or by radioactive labeling. o Immunological detection Antibodies can be obtained that bind specifically to the target Label antibodies with a detection method Photons generated by chemical reaction (color) Radioactive label o Subcellular fractionation Isolate organelles Nuclei Mitochondria or chloroplasts Small membrane fragments – golgi, ER, plasma membrane Soluble phase – cytosol Accomplished by differential centrifugation Heavier components move faster through solution when subjected to centripetal force o Differential precipitation When ion concentrations raise the amount of water available to the proteins decreases Proteins precipitate Different proteins precipitate at different salt concentrations Add salt gradually, collecting precipitated proteins at each step Remove salt by dialysis Membrane permeable to small molecules but not proteins o Chromatographic Separation – Gel Filtration Drip protein mixture in solution through column filled with inert mesh beads Various mesh sizes available Small molecules enter mesh, large molecules excluded Less effective volume for larger molecules, so they elute first Collect fractions while solution flows Identify fractions containing target o Enzyme assay o Immunodetection o Chromatographic separation – Ion Exchange Separation based on net change At different pH, proteins have different charge based on its amino acid sequence Flow protein solution through inert matrix decorated with charged functional groups Oppositely charged proteins will bind, same like-charged proteins flow through Apply increasing concentrations of NaCl salt ions neutralize charges on matrix proteins detach and elute Different proteins elute at different salt concentrations, depending on charge o Chromatographic Separation – Affinity Selection Flow protein mixture in solution through column containing inert beads covalently bound to a specific ligand Proteins that can bind that to the ligand will stick to the column, others flow through Wash extensively with buffer Add free ligand in high concentration Protein binds to free ligand rather than immobilized ligand Elutes from column Most often used with recombinant proteins o Polyacrylamide Gel Electrophoresis Allows visualization of protein Proteins denatured by boiling and binding to sodium dodecyl sulfate (SDS) Alkane portion of SDS binds to peptide backbone Sulfate group provides negative charge o Constant charge/mass ration SDS-protein complexes move through gel in electric field Smaller proteins move faster through the gel Stain with general protein stain, Coomassie Blue Quantification of Purification o At each step determine: Enzyme activity units Total protein Purification activity/total protein o Calculate yield and fold-purification Immunological Detection of Proteins o Antibodies – proteins produced by cells in mammalian blood Millions of possible variable structures Presence of foreign protein (antigens) stimulates production of antibodies that bind to that protein o Synthesize small peptide antigen from a known protein sequence Inject a rabbit, wait several weeks, collect blood serum Antibodies that bind that antigen present in serum Purify these antibodies Antibody reagent Immunoprecipitation o Antibodies can be used as affinity purification ligands Antibody is bound to inert bead Suspend beads in solution or pack them into a column Immunoblot to detect proteins (Western blot) o SDS-PAGE separates proteins in purification fraction o Add primary antibody o Add labeled secondary antibody o Detect labeled secondary antibody Expose to film Strength of signal indicates quantity of target protein present Enzyme-linked Immunosorbent Assay (ELISA) o Can be used to detect presence of antibodies in serum Ex HIV infection Attach antigen to bottom of well in plastic plate (large excess) Expose to serum Antibodies bind to antigen, if they are present Add secondary antibody that binds to any human antibody Conjugated to detection enzyme Count photons emitted Proportional to amount of antibody present Sandwich ELISA (capture ELISA) o Used to quantify antigen (protein) in solution Attach specific antibody #1 to bottom of well Expose to solution to be tested Antigen binds antibody #1 Expose to specific antibody #2, which binds antigen protein at a different location Expose to antibody #3 General binding to ANY mouse antibody, in constant region Enzyme conjugated to generate photons and quantify Determination of Amino Acid Sequence of a protein o Mass spectrometry provides amino acid sequence of small fragments of the protein o Compare to all predicted sequences possible in the organism Known from genome sequence o Utility Evolutionary comparisons Disease states Subcellular location Identify gene, obtain mutations
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