Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods.
New NMR experiments are presented for the assignment of methyl (13)C and (1)H chemical shifts from Ile, Leu, and Val residues in high molecular weight proteins. The first class of pulse schemes transfers magnetization from the methyl group to the backbone amide spins for detection, while the second more sensitive class uses an "out-and-back" transfer scheme in which side-chain carbons or backbone carbonyls are correlated with methyl (13)C and (1)H spins. Both groups of experiments benefit from a new isotopic labeling scheme for protonation of Leu and Val methyl groups in large deuterated proteins. The approach makes use of alpha-ketoisovalerate that is (13)C-labeled and protonated in one of its methyl groups ((13)CH(3)), while the other methyl is (12)CD(3). The use of this biosynthetic precursor leads to production of Leu and Val residues that are (13)CH(3)-labeled at only a single methyl position. Although this labeling pattern effectively reduces by 2-fold the concentration of Leu and Val methyls in NMR samples, it ensures linearity of Val and Leu side-chain (13)C spin-systems, leading to higher sensitivity and, for certain classes of experiments, substantial simplification of NMR spectra. Very near complete assignments of the 276 Ile (delta 1 only), Leu, and Val methyl groups in the single-chain 723-residue enzyme malate synthase G (MSG, molecular tumbling time 37 +/- 2 ns at 37 degrees C) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments.
Contribution from the Protein Engineering Network Centres of Excellence and the Departments of Medical Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Vitali
V
Tugarinov
Lewis E
LE
Kay
D002247:Carbon Isotopes; D007532:Isoleucine / Q000737:chemistry*; D007930:Leucine / Q000737:chemistry*; D008292:Malate Synthase / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011522:Protons; D014633:Valine / Q000737:chemistry*
D002247:Carbon Isotopes; D011522:Protons; D007532:Isoleucine; D008292:Malate Synthase; D007930:Leucine; D014633:Valine