A sample was used in which half of the pfLamA molecules had their calcium-binding site filled with Ca2+.

Identical amino acid sequences were used in the X-ray and NMR investigation. In case of the X-ray study all molecules contained Ca2+.

Information obtained from PDB's Chemical Component Dictionary
at http://wwpdb-remediation.rutgers.edu/downloads.html
Downloaded on Tue Jun 9 13:21:54 2009

1H proton chemical shifts are referenced to internal DSS.

Indirect chemical shift referencing of 15N and 13C (relative to 1H DSS set to 0
ppm) and recalibration to a temperature of T=62 oC (T=335 K). The temperature of
the NMR sample in the probe is derived from the following temperature
calibration samples: a sample of DSS in 90%/10% H2O/D2O and a sample of neat
MeOH.

The temperature corrections for the indirect chemical shift ratios of 13C and
15N are done according to the temperature correction given in:

Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield,
E., Markley, J. L., and Sykes, B. D., "1H, 13C and 15N Chemical Shift
Referencing in Biomolecular NMR," J. Biomol. NMR 6, 135-140 (1995).

The indirect chemical shift ratios used at 62 oC are:
15N (0 ppm DSS)= 0.101329128
13C (0 ppm DSS)= 0.251449567

Chemical shifts of 15N and 13C are therefore +0.097 ppm and +0.148 ppm,
respectively, compared to standard IUPAC indirect chemical shift referencing
rules.

Biochemical and biophysical research communications
NMR characterization of a 264-residue hyperthermostable endo-beta-1,3-glucanase.

Calculated mass (natural abundance): 30085 Dalton.
Exptl mass by MALDITOF: 30135 Dalton (including calcium ion and with oxidized
Met34).

The N-terminal amino acid Q34, originally present in the full-length pfLamA
sequence, is substituted for Met34 in the pfLamA(34-297) protein construct
studied by NMR spectroscopy & crystallography. According to mass spectrometry,
in about 10% of the pfLamA molecules Met34 is oxidized.

Sequence numbers corresponds to the native pfLamA sequence without signal peptide.

Amides of amino acid residues that are in proximity to the calcium-binding site
cause the presence of two sets of 1H and 15N NMR signals, as the protein is in
slow exchange between calcium-containing and non calcium-containing
conformations.

hydrolysis of beta-1,3 linked glucose polymers (laminarin)

The X-Ray Structure Of Endo-Beta-1,3-Glucanase From Pyrococcus Furiosus

endo-beta-1,3-glucanase precursor [Pyrococcus furiosus DSM 3638]

endo-beta-1,3-glucanase [Pyrococcus furiosus DSM 3638]

endo-beta-1,3-glucanase [Pyrococcus furiosus COM1]

endo-beta-1,3-glucanase [Pyrococcus furiosus]

1H, 13C and 15N resonance assignments of the hyperthermostable 264-residue endo-<beta>-1,3-glucanase LamA at 62 C

A sample was used in which half of the uniformly 15N/13C-labelled pfLamA
molecules had their calcium-binding site filled with Ca2+.

A sample was used in which all uniformly 15N/13C-labelled pfLamA molecules had
their calcium-binding site filled with Ca2+ (achieved by adding 10 mM CaCl2 to a
sample of protein that contains no calcium).

A sample was used in which half of the non-labelled pfLamA molecules had their
calcium-binding site filled with Ca2+.

A sample was used in which half of the uniformly 15N-labelled pfLamA molecules
had their calcium-binding site filled with Ca2+.

High temperature: T = 335 K (i.e., 62 oC).
Error in temperature is maximally 1 K, as estimated from an additional NMR
sample containing buffer and DSS. The difference in chemical shift between H2O
and DSS was plotted as a function of temperature. Temperature was determined
using an internal thermocouple in the NMR tube. Subsequently, the difference in
chemical shift between H2O and DSS of the pfLamA sample led to the determination
of the actual temperature of the protein sample.

Software for automated backbone assignment of proteins

Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax

Department of Biochemistry, Duke University Medical Center, Durham, NC, USA