The conformational properties of the basic pancreatic trypsin-inhibitor have been studied using both physical and chemical techniques.The structural properties of this mini-protein are quite unusual. The folded conformation remains unaltered a t 77 "C and pH 2.1 or in 6 M guanidine-HCI at pH 7.5. The selective cleavage [22] of the disulfide bridge Cys,, -Cys,, reduces considerably the stability of the inhibitor towards heat or guanidine-HC1. A conformational study of the carboxamidomethyl, carboxymethyl, and aminoethyl derivatives of the inhibitor has been carried out after partial reduction and modification of Cys14 and Cys,, by iodoacetamide, iodoacetic acid, and ethyleneimine.There exist three folded isomeric forms of the inhibitor between pH 1 and 11. Form I is the most stable form; it exists near neutral pH. It is extremely thermostable and remains folded in 6 M guanidine-HC1. Form I1 is stable below pH 1.5-2; its transition temperature is 81 "C. Form I11 is predominant at pH 10-11 at 20 "C. A detailed analysis of the conformational isomers I, I1 and I11 has been ca,rried out with the carboxamidomethylated inhibitor.Both physico-chemical and chemical data indicate that the acidic conformational change I + I1 is dependent upon the ionisation of a buried carboxylate which may be the side-chainThe alkaline isomerisation I + I11 is induced by the unmasking of the a-ammonium of the N-terminal arginine residue. The apparent pK of the group is 9.4 a t 25 "C. Acetylation of the a-amino group stabilizes a form very similar to I11 and prevents the formation of form I a t neutral pH.of Glu,.The amino-acid sequence of the basic trypsininhibitor from bovine pancreas is known [l --31 and the structure-function properties of the protein have been studied in several laboratories [4-141. Lysine-15 was shown to be an essential element of the active site [lo].Since this trypsin inhibitor is comprised of only 58 amino acids in a single polypeptide chain it is one of the smallest known proteins. It is consequently an excellent model for the description of the structural properties of mini-proteins. Others studies on miniproteins have been carried out in this laboratory with neurotoxins composed of 63 amino acids [15].The inhibitor reacts with trypsin, one of the best known enzymes [16,17], to form an extremely stable 1 : 1 complex [18,19]. Because of the considerable structural as well as functional knowledge concerning the two partners in the complex and because of the very unusual stability of their association, the interaction between trypsin and its inhibitor appears t o be an excellent model system for the study of effector-receptor association in the protein field.
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