Lecture5 - ecture 5 10/3/11 utline: Amino acid sequence...

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Unformatted text preview: ecture 5 10/3/11 utline: Amino acid sequence determination: Amino-terminal amino acid determination. Carboxyl-terminal amino acid determination. Specific fragmentation of peptides and proteins. Edman degradation. Determination of amino acid sequences from genomic or complementary DNA. Functional diversity of proteins. Primary structure of proteins. Secondary structure of proteins. X-ray crystallography. ackground Reading: Berg, et al.: pages 38 45 pages 80 84 pages 98 - 100 Garrett and Grisham: pages 109 110 pages 116 -123 Assignment: Segel Pages: 141 142: Problems 1, 2, 3 and 5 Amino acid sequence determination. The first step in the determination of the amino acid sequence of a peptide or protein is to isolate it and determine its amino acid composition. In the last lecture we examined how to determine the amino acid composition. The next step is to determine the amino acid present at the N-terminus and C-terminus of the peptides. Amino-terminal amino acid determination Either of two reagents are commonly used. Both react with free NH 2 groups. 1) Sanger reagent 2,4-fluorodinitrobenzene (FDNB) DNP-amino acid After reaction, hydrolyze the peptide and will obtain the DNP -amino acid as well as DNP-lysine where the DNP is on the epsilon amino group. Micromolar sensitivity 2) Dansyl chloride. Dansyl amino acid Fluorescent product. This reagent also reacts with both the -amino group as well as the epsilon amino group of lysine. Nanomolar sensitivity. Carboxyl-terminal amino acid determination Usually conducted by using an enzyme that can specifically remove the COOH-terminal amino acid. Carboxypeptidase A is an exopeptidase that can remove all amino acids except lysine, arginine or proline and will not remove any amino acid if proline is the penultimate amino acid (second from the right). Carboxypeptidase B is an exopeptidase that will only remove lysine or arginine. Carboxypeptidase Y is an exopeptidase that can remove any amino acid from the COOH-terminal end. Specific fragmentation of peptides and proteins Enzymatic methods: Trypsin is an endoprotease that specifically hydrolyzes the peptide bond at the carbonyl end of lysine or arginine....
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This note was uploaded on 01/15/2012 for the course BIS 102 taught by Professor Hilt during the Fall '08 term at UC Davis.

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Lecture5 - ecture 5 10/3/11 utline: Amino acid sequence...

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