title: Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy.
abstract: Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.
authors.affiliation: Department of Biochemistry and Molecular Biology, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Brockerman
authors.forename: Mark
authors.initials: M
authors.lastname: Okon
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D012694:Serine / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*
chemical_list: D011522:Protons; D013912:Threonine; D012694:Serine; D043364:Endo-1,4-beta Xylanases