BCHE 395-6 2013 Protein Motifs and Sequencing

loss of biological ac1vity increase reac1vity of

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Unformatted text preview: 1cs, Height is degree of conserva1on 17 Primary Structure Determina1on Chapt. 3 p. 97 start •  Protein “sequencing” •  1953: Frederick Sanger à༎ sequenced insulin •  Used to ID proteins –  More frequent prior to gene sequencing explosion –  S1ll necessary for confirma1on –  The principles of the methods are s1ll commonly employed for different purposes 18 Methods for I° Structure Determina1on •  Genng the protein ready: –  (1) Complete protein denatura1on •  (I° structure intact!) –  (2) Cleave disulfide linkages •  Then: –  (3) N- terminal sequencing •  Amide bond targeted –  (4) Pep1de fragmenta1on and sequencing •  Amide bond targeted 19 Methods for I° Structure Determina1on 1.  Complete protein denatura1on (loss of II° and III° (& IV°) hKp://food.drricky.net/2012/10/ cooking- with- acid.html a.  Heat (Cooking. Duh.) b.  Strong acid or base treatments (Stomach acid?) −COO- Ÿ༉Ÿ༉Ÿ༉+H3N− à༎ −COOH +H3N− c.  Freeze/thaw cycles (H2O disrup1on) d.  Organic solvents - hydrophobic and H- bonding e.  Special reagents - urea, guanidine HCl, SDS detergent 20 Protei...
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