Figure 1.
Metabolism of Thr starting from Asp.
The structure of Thr is highlighted in the box and the Thr to Asp pathway is along the vertical. Also shown are pathways that potentially can be exploited to synthesize Thr and the enzymes that are involved (horizontal). Dashed lines (bottom) indicate the non-physiological conversion of 2-ketobutyrate into L-2-aminobutyrate and then into Thr.
Figure 2.
Scheme used for the biosynthesis of U-[2H],Thr-γ2[13CH3], 6.
Enzyme name abbreviations: KHGA = 2-keto-4-hydroxyglutarate aldolase, AAT = aspartate aminotransferase, HSK = homoserine kinase, TS = threonine synthase, BCAT = branched-chain-amino-acid aminotransferase.
Figure 3.
NMR spectra of L-[α-2H;β−2H;γ-13C]-Thr, 6.
(A) 1H NMR spectrum with vertical scale adjusted to fit entire methyl doublet. (B) Same spectrum as in A with a 50-fold enhanced vertical scale. (C) 13C,1H HSQC spectrum of the same sample, 6. Contour level is set to 0.5% of the Thr methyl signal. The only 3 other peaks (all under 1% of the intensity of the Thr methyl) derive from impurities and are indicated with arrowheads. Approximately 2% residual protonation remains at the α position and much less at β.
Figure 4.
Efficacy of Thr labeling in proteins.
Fraction of 13C intensity lost at Thr Cγ (A), Ile Cδ1 (B) and Glu Cγ (C) positions of a U-[13C,1H] labeled Abp1p SH3 domain sample where 12C labeled precursors such as Thr, 2-ketobutyrate or 2-ketoisovalerate (2KIV) are added in the amounts indicated at the bottom of the figure prior to induction of protein expression. The value of ‘n’ corresponds to the number of residues of a particular type that are averaged over in each measurement, with the error bars denoting the complete range of values obtained. Details are in Supporting Information S1.
Figure 5.
NMR spectroscopy of Thr-γ2[13CH3]-labeled proteins.
(A) 13C,1H HMQC spectrum of U-[2H] α7β7β7α7 prepared with U-[13C,1H]-Thr (labeled in the β subunit, 200 µM subunit concentration) recorded in 18 hours, 70°C, 18.8T. 13Cβ Thr band selective adiabatic decoupling [65] was applied to narrow the Thr 13Cγ resonance lines. (B) 13C,1H HMQC spectrum of 0.54 mM (subunit concentration) U-[2H], Ile-δ1[13CH3], Thr-γ2[13CH3] α7β7β7α7 (labeled in the β subunit) recorded in 2 hours at 70°C, 18.8T. Partial assignments are indicated; stars denote degradation product peaks. The difference in acquisition times is intended to compensate for the concentration differences. It is clear, however, that much higher quality spectra are derived from samples prepared with the current labeling scheme.