Design of hydropathically complementary peptides for Big Endothelin affinity purification.
Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH2-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.
Fassina
G
G
Protein Engineering Unit, TECNOGEN S.c.p.A., Milan, Italy.
Consonni
R
R
Zetta
L
L
Cassani
G
G
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019332:Endothelin-1; D016232:Endothelins / Q000737:chemistry / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis*; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification*
D019332:Endothelin-1; D016232:Endothelins; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors