Identification of a Residue Critical for Maintaining the Functional Conformation of BPTI

• Published in: Journal of molecular biology Vol. 333; no. 2; pp. 425 - 441
• Main Authors: Miachel Hanson, W., Beeser, Scott A., Oas, Terrence G., Goldenberg, David P.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 17.10.2003
• Subjects: Animals; Aprotinin - chemistry; Aprotinin - genetics; Aprotinin - metabolism; backbone dynamics; Cattle; Crystallography, X-Ray; Lipari-Szabo mapping; Magnetic Resonance Spectroscopy - methods; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; NMR relaxation; protease inhibitor; Protein Conformation; Protein Folding; spectral density functions; Trypsin - metabolism; Trypsin Inhibitors - metabolism; Tyrosine - chemistry; TOCSY, total coherence spectroscopy; HSQC, heteronuclear single quantum coherence; spectral density functions; protease inhibitor; backbone dynamics; BPTI, bovine pancreatic trypsin inhibitor, amino acid replacements are indicated by the wild-type residue type (using the one letter code for the 20 standard amino acid residues), followed by the residue number and the mutant residue type; NMR, nuclear magnetic resonance; Lipari-Szabo mapping; NOESY, nuclear Overhauser effect spectroscopy; NMR relaxation; NOE, nuclear Overhauser effect
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2003.08.023

The effects of amino acid replacements on the backbone dynamics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments. Previous studies have shown that backbone amide groups within the trypsin-binding region of the wild-type protein undergo conformational exchange processes on the μs time scale, and that replacement of Tyr35 with Gly greatly increases the number of backbone atoms involved in such motions. In order to determine whether these mutational effects are specific to the replacement of this residue with Gly, six additional replacements were examined in the present study. In two of these, Tyr35 was replaced with either Ala or Leu, and the other four were single replacements of Tyr23, Phe33, Asn43 or Asn44, all of which are highly buried in the native structure and conserved in homologous proteins. The Y35A and Y35L mutants displayed dynamic properties very similar to those of the Y35G mutant, with the backbone segments including residues 10–19 and 32–44 undergoing motions revealed by enhanced 15N transverse relaxation rates. On the other hand, the Y23L, N43G and N44A substitutions caused almost no detectable changes in backbone dynamics, on either the ns–ps or ms–μs time scales, even though each of these replacements significantly destabilizes the native conformation. Replacement of Phe33 with Leu caused intermediate effects, with several residues that have previously been implicated in motions in the wild-type protein displaying enhanced transverse relaxation rates. These results demonstrate that destabilizing amino acid replacements can be accommodated in a native protein with dramatically different effects on conformational dynamics and that Tyr35 plays a particularly important role in defining the conformation of the trypsin-binding site of BPTI.