Work

IA3, A Yeast Proteinase A Inhibitor, Is Intrinsically Unstructured in Solution

Year: 2002

Type: article

Abstract: INTRODUCTION.IA 3 is a 68 amino acid yeast protein that inhibits yeast proteinase A (YprA) with a K i of about 1 nM.No other aspartic proteinase has been found to be inhibited by IA 3 , and at least 15 aspartic proteinases related to YprA cleave IA 3 as a substrate [1,2].An X-RAY crystal structure has shown that the first 34 amino acids of IA 3 binds to YprA as an α-helix[3].IA 3 does not crystallize in the absence of YprA, and we have now found by NMR and CD that IA 3 is unfolded in solution and undergoes a large structural transition upon binding to YprA. METHODS.Recombinant IA 3 with a His-tag for purification was produced in E. coli with 15 N or 15 N/ 13 C uniform isotopic enrichment.Triple resonance NMR was used to assign most of the backbone and side-chain resonances of free 15 N/ 13 C-labeled IA 3 .Samples of 15 N-labeled IA 3 were then added to natural abundance YprA and were titrated with YprA and TFE (trifluoroethanol).Circular dichroism (CD) was also used to monitor changes of IA 3 upon addition of TFE.The inhibition constants (K i ) of IA 3 for YprA were determined as a function of temperature for van't Hoff analysis. RESULTS.The chemical shifts of free IA 3 were found to be essentially random-coil values.The CD spectrum of IA 3 was characteristic of a random-coil peptide but changed to α-helix upon addition of approximately 30% TFE.The 15 N-HSQC NMR spectra of IA 3 were characteristic of random-coil structure, but the addition of YprA produced a large structural transition in IA 3 (Fig. 1).The IA 3 samples used for NMR analysis were active and inhibited YprA with a K i of about 2 nM. 1 H-15 N heteronuclear NOEs at 600 MHz of free IA 3 were very small or negative, consistent with the expected dynamics of an unfolded protein.A van't Hoff analysis of the temperaturedependence of the Ki of IA 3 for YprA was nonlinear, suggesting a rather complex coupled equilibria of protein folding and inhibition (Fig. 2).

Source: The Scientific World JOURNAL

DOI: 10.1100/tsw.2002.47