Journal of Biomolecular NMR, 13: 369–374, 1999. KLUWER/ESCOM © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 369 A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15 N-, 13 C-, 2 H-labeled proteins Natalie K. Goto a,b , Kevin H. Gardner c,∗ , Geoffrey A. Mueller b,c , Randall C. Willis b & Lewis E. Kay c a Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 b Department of Biochemistry and Structural Biology, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5S 1X8 c Protein Engineering Network Centres of Excellence and Departments of Medical Genetics and Microbiology, Biochemistry and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 Received 8 December 1998; Accepted 15 January 1999 Key words: deuteration, α-ketobutyrate, α-ketoisovalerate, methyl protonation Abstract A selective protonation strategy is described that uses [3- 2 H] 13 C α-ketoisovalerate to introduce ( 1 H-δ methyl)- leucine and ( 1 H-γ methyl)-valine into 15 N-, 13 C-, 2 H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≈100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3- 2 H 2 ] α-ketobutyrate to the expression media (D 2 O solvent) results in the production of proteins with ( 1 H-δ1 methyl)-isoleucine (>90% incorporation). 1 H- 13 C HSQC correlation spectroscopy establishes that CH 2 D and CHD 2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective. The application of multidimensional NMR spec- troscopy to study the structure and dynamics of macro- molecules requires techniques that maximize both spectral resolution and sensitivity. To this end, uni- form labeling of molecules with NMR-active isotopes, combined with the development of triple-resonance experiments has permitted detailed NMR analyses of systems up to 20 kDa in molecular mass (Bax, 1994). Beyond this limit, spectra become increasingly com- plicated by peak overlap, while shorter transverse relaxation times lead to decreases in both sensitivity and resolution. In this regard deuteration of aliphatic carbons is increasingly used as a means of spectral ∗ Present address: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9038, U.S.A. Supporting information available: One table showing per- cent incorporation versus α-ketoisovalerate added obtained from GC-MS (with errors at each value of added α- ketoisovalerate). This material can be obtained by e-mailing N.K.G. (gnat@pound.med.utoronto.ca). Figure 1. Chemical structures of metabolites supplementing the 13 C, 2 H-glucose E. coli growth media (D 2 O) used in the selective protonation strategy described in the text and the resulting amino acid products produced (Gottschalk, 1986). Carbons derived from each of the metabolite precursors are indicated by ∗ .