Characterization of mutant xylanases using Fourier transform ion cyclotron resonance mass spectrometry: Stabilizing contributions of disulfide bridges and N-terminal extensions
UNSPECIFIED. (2004) Characterization of mutant xylanases using Fourier transform ion cyclotron resonance mass spectrometry: Stabilizing contributions of disulfide bridges and N-terminal extensions. BIOCHEMISTRY, 43 (29). pp. 9556-9566. ISSN 0006-2960Full text not available from this repository.
Official URL: http://dx.doi.org/10.1021/bi049597b
Structural properties and thermal stability of Trichoderma reesei endo-1,4-beta-xylanase II (TRX 11) 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 Il 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 11, DS2, DS5, and 1313 1 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 degreesC 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 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 similar to5 degreesC.
|Item Type:||Journal Article|
|Subjects:||Q Science > QD Chemistry|
|Journal or Publication Title:||BIOCHEMISTRY|
|Publisher:||AMER CHEMICAL SOC|
|Date:||27 July 2004|
|Number of Pages:||11|
|Page Range:||pp. 9556-9566|
Actions (login required)