Faculty Collaboration Database

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy. Biochemistry 2001 May 29;40(21):6275-83

Date

05/24/2001

Pubmed ID

11371189

DOI

10.1021/bi0100992

Scopus ID

2-s2.0-0035967533 (requires institutional sign-in at Scopus site)   34 Citations

Abstract

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.

Author List

Peterson FC, Gordon NC, Gettins PG

Author

Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Alanine
Animals
Aprotinin
Aspartic Acid
Cattle
Enzyme Activation
Escherichia coli
Macromolecular Substances
Mutagenesis, Site-Directed
Nitrogen Isotopes
Nuclear Magnetic Resonance, Biomolecular
Protein Folding
Protons
Serine
Trypsin
Trypsin Inhibitor, Kunitz Soybean
Trypsin Inhibitors
Trypsinogen