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Unambiguous determination of the ionization state of a glycoside hydrolase active site lysine by 1H-15N heteronuclear correlation spectroscopy


, : Unambiguous determination of the ionization state of a glycoside hydrolase active site lysine by 1H-15N heteronuclear correlation spectroscopy. Journal of the American Chemical Society 128(48): 15388-9

We have investigated the lysine side chain amines in the 34 kDa catalytic domain from Cellulomonas fimi beta-(1,4)-glycosidase Cex (or CfXyn10A) using 1H-detected 15N heteronuclear correlation NMR spectroscopy. Signals from the 1Hzeta ( approximately 8 ppm) and 15Nzeta ( approximately 35 ppm) of Lys302 in the unmodified enzyme and Lys47 in a trapped cellobiosyl-enzyme intermediate were detected in a 1H-15N HMQC spectrum (pH 6.5 and 30 degrees C). The amine of Lys302 forms a buried ion pair, and that of Lys47 is hydrogen bonded to the cellobioside. Both lysines are positively charged, as unambiguously demonstrated by the splitting of their 15Nzeta signals into quartets (|1JNH| approximately 75 Hz) in a 1H-15N HSQC spectrum recorded without 1H decoupling during 15N evolution. Qualitative insights into the dynamic properties of these lysines are also provided by the deviations of their quartet intensity ratios from that of approximately 3:1:1:3 expected for a highly mobile amine. On the basis of the observed ratios of approximately 1:1:1:1 for the quartet of Lys302 and approximately 0.5:1:1:0.5 for Lys47, the amine of the latter active site residue is most rigidly positioned. Signals from at least 8 and 10 additional positively charged, mobile amines in Cex were observed at 10 degrees C and pH 6.5 and 5.6, respectively. By using conditions of reduced temperature, slightly acidic pH, and low general base concentrations, as well as water flipback pulses to minimize the effects of hydrogen exchange, 1H-15N correlation experiments provide a sensitive route to directly investigate the charge states and dynamic properties of the N-terminal and side chain amines in proteins and protein complexes.

US$19.90

PMID: 17132001

DOI: 10.1021/ja065766z


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de Aza, P.N.; Zuleta, F.; Velasquez, P.; Vicente-Salar, N.; Reig, J.A., 2014: (α'(H))-Dicalcium silicate bone cement doped with tricalcium phosphate: characterization, bioactivity and biocompatibility. The influence of phosphorus doping on the properties of (α'(H))-dicalcium silicate (C(2)S) bone cement was analyzed, in addition to bioactivity and biocompatibility. All the cements were composed of a solid solution of TCP in C(2)S ([Formula...

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Gerbermann, H., 1989: Current situation and alternatives for diagnosis and control of chlamydiosis in the Federal Republic of Germany. After a description of import and quarantine regulations for psittacines in the GFR and antibiotic treatment, the development of a specific, sensitive and rapid serological test for avian chlamydiosis is described. A commercial amplified ELISA for...

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