lecture_6 - Structural hierarchy in proteins Color...

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Structural hierarchy in proteins
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Color conventions
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Protein Geometry CORN LAW amino acid with L configuration
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Greek alphabet
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The Polypeptide Chain
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Chapter 5 Covalent structures of proteins Proteins function as: 1. Enzymes:biological catalysts 2. Regulators of catalysis-hormones 3. Transport and store i.e. O , metal ions
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6. Cellular defense immuoglobulins Antibodies Killer T cell Receptors 7. Structural Collagen Silk, etc. Function is dictated by protein structure!!
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There are four levels of protein structure 1. Primary structure 1 ° = Amino acid sequence, the linear order of AA’s. Remember from the N-terminus to the C-terminus Above all else this dictates the structure and function of the protein.
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There are four levels of protein structure 2. Secondary structure 2 ° = Local spatial alignment of amino acids without regard to side chains. Usually repeated structures Examples: α helix, β sheets, random coil, or β turns
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3. Tertiary Structure 3 ° = the 3 dimensional structure of an entire peptide. Great in detail but vague to generalize. Can reveal the detailed chemical mechanisms of an enzyme.
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4. Quaternary Structure 4 ° two or more peptide chains associated with a protein. Spatial arrangements of subunits. Chapter 5.3 is how to determine a protein’s primary structure. “Protein Chemistry
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Example of each level of protein structure
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Insulin was the first protein to be sequenced F. Sanger won the Nobel prize for protein sequencing. It took 10 years, many people, and it took 100 g of protein! Today it takes one person several days to sequence the same insulin. 1021 AA β- glactosidase 1978
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Steps towards protein sequencing Above all else, purify it first!! Chapter 5.3 then 5.1 and 5.2 1. Prepare protein for sequencing a. Determine number of chemically different polypeptides. b. Cleave the protein’s disulfide bonds. c. Separate and purify each subunit. d. Determine amino acid composition for each peptide.
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Bovine insulin: note the intra- and inter- chain disulfide linkages
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2. Sequencing the peptide chains: a. Fragment subunits into smaller peptides 50 AA’s in length. b. Separate and purify the fragments c. Determine the sequence of each fragment. d. Repeat step 2 with different fragmentation system.
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3. Organize the completed structure. a. Span cleavage points between sets of peptides determined by each peptide sequence. b. Elucidate disulfide bonds and modified amino acids. At best, the automated instruments can sequence about 50 amino acids in one run! Proteins must be cleaved into smaller pieces to obtain a complete sequence.
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How many peptides in protein? Bovine insulin should give 2 N-terminii and 2 C- terminii N-terminus 1-Dimethylamino - naphthalene-5-sulfonyl chloride Dansyl chloride Reacts with amines: N-terminus + Lys (K) side chains End Group Analysis
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use 6M HCl to cleave off the derivatized amino acid, this also cleaves all other amide bonds (residues) as well. Edman degradation with Phenyl isothiocyanate, PITC
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