Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis.
Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.
Ebina
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Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Lerner
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Wright
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D000107:Acetylation; D007611:Aprotinin / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002918:Chymotrypsin / Q000378:metabolism; D003432:Cross-Linking Reagents; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008782:Methylurea Compounds; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D013388:Succinimides; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D006146:Guanidines; D008782:Methylurea Compounds; D010455:Peptides; D013388:Succinimides; C002249:O-methylisourea; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D007611:Aprotinin; D002918:Chymotrypsin; D014357:Trypsin; D019791:Guanidine; D008239:Lysine; D003545:Cysteine; C001426:N-hydroxysuccinimide; D004134:Dimyristoylphosphatidylcholine