1 | 600 uM [U-13C; U-15N] Tpt1, 2.4 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2O | 4-fold excess of NAD+ was used to assure full saturation of Tpt1 | CN_sample195% H2O/5% D2Osolution2 | 280 uM [U-13C; U-15N] Tpt1, 1.12 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2O | 4-fold excess of NAD+ was used to assure full saturation of Tpt1 | CN_sample295% H2O/5% D2Osolution3 | 325 uM [U-13C; U-15N] Tpt1, 1.3 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 100% D2O | 4-fold excess of NAD+ was used to assure full saturation of Tpt1 | CN_sample3100% D2Osolution4 | 300 uM [U-13C; U-15N] Tpt1, 1.2 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 100% D2O | Tpt1 was uniformly [2H-15N] label with specific [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2). 4-fold excess of NAD+ was used to assure full saturation of Tpt1 | 2H-15N_ILVM_sample4_d2O | 100% D2Osolution5 | 500 uM [U-13C; U-15N; U-2H] Tpt1, 2 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 95% H2O/5% D2O | Tpt1 was uniformly [2H-15N] label with specific [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2). 4-fold excess of NAD+ was used to assure full saturation of Tpt1 | DCN_sample595% H2O/5% D2Osolution6 | 400 uM [U-13C; U-15N] Tpt1, 1.6 mM NICOTINAMIDE-ADENINE-DINUCLEOTIDE, 20 mM HEPES, 200 mM sodium chloride, 2 mM DTT, 2 mM [U-2H] EDTA, 5 % v/v [U-2H] glycerol, 1 mM AEBSF protease inhibitor, 0.04 % w/v sodium azide, 50 uM DSS, 100% D2O | Tpt1 was uniformly [2H-15N] label with specific [13C-1H] labeling on methyl groups for ILE [d1 only] VAL (g1 and g2), Leu (d1 and d2). 4-fold excess of NAD+ was used to assure full saturation of Tpt1 | 2H-15N_ILVM_sample5_d2O | 100% D2O | | | | | | | | | | | | | | |