Characterization of mutant xylanases using fourier transform ion cyclotron resonance mass spectrometry: stabilizing contributions of disulfide bridges and N-terminal extensions.
Structural properties and thermal stability of Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) and its three recombinant mutants were characterized using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry and hydrogen/deuterium (H/D) exchange reactions. TRX II has been previously stabilized by a disulfide bridge C110-C154 and other site-directed mutations (TRX II mutants DS2 and DS5). Very recently, a highly thermostable mutant was introduced by combining mutations of DS5 with an N-terminal disulfide bridge C2-C28 (mutant DB1). Accurate mass measurements of TRX II, DS2, DS5, and DB1 verified the expected DNA-encoded protein sequences (average mass error 1.3 ppm) and allowed unequivocal assignment of the disulfides without chemical reduction and subsequent alkylation of the expected cross-links. Moreover, H/D exchange reactions provided means for the detection of a major heat-induced conformational change comprising two interconverting conformers of very different H/D exchange rates as well as allowed the apparent melting temperatures (T(m)) to be determined (62.6, 65.1, 68.0, and 82.2 degrees C for TRX II, DS2, DS5, and DB1, respectively). Residual activity measurements verified that the enzymes inactivated at significantly lower temperatures than expected on the basis of the apparent T(m) values, strongly suggesting that the inactivation takes place through minor conformational change other than observed by H/D exchange. ESI FT-ICR analyses also revealed molecular heterogeneity in DS5 and DB1 due to the propeptide incorporation. Resulting unintentional N-terminal extensions were observed to further improve the stability of the DB1 mutant. The extension of six amino acid residues upstream from the protein N-terminus increased stability by approximately 5 degrees C.
Jänis
Janne
J
Department of Chemistry, University of Joensuu, P.O. Box 111, FI-80101 Joensuu, Finland.
Turunen
Ossi
O
Leisola
Matti
M
Derrick
Peter J
PJ
Rouvinen
Juha
J
Vainiotalo
Pirjo
P
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D005583:Fourier Analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D014995:Xylosidases