Application Note 48

Stereospecific Leu/Val Methyl Labeling: An Important Technology for NMR Studies of High-Molecular-Weight Complexes

Rui Huang¹ and Lewis E. Kay^1,2

1. Departments of Molecular Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
2. Hospital for Sick Children, Program in Molecular Medicine, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada

The development of modern biomolecular solution NMR spectroscopy has paralleled innovations in labeling technologies. For example, early advances in heteronuclear double- and triple-resonance NMR spectroscopy were closely coupled to the emergence of methodologies for the effective uniform labeling of biomolecules with ¹⁵N and/or ¹³C.^1,2 Despite arguments that uniform ¹³C (U-¹³C) labeling would be prohibitively expensive, the high demand for U-¹³C-labeled precursors has significantly lowered costs, and the preparation of biomolecules with ¹³C incorporation is most often the approach of choice for NMR studies.³ As the size of the system studied increases, both the sensitivity and the resolution of the resulting spectra suffer, and this has led to the development of amide-TROSY-based experiments⁴ that are significantly improved through the use of perdeuteration.⁵ A powerful labeling scheme for studies of proteins over a wide range of molecular masses, but, in particular, for systems with aggregate masses in excess of 100 kDa involves ¹³CH₃-labeling in a highly deuterated background.⁶ Initial schemes focused on Ile (δ1), Leu and Val methyl groups, where for Leu and Val only one of the isopropyl methyls is ¹³CH₃ (the other is ¹²CD₃), but more recently precursors for the other methyl positions have become commercially available^7-11 so that it is possible to label the methyl group of choice. Once again, the availability of precursors stimulated the development of new NMR experiments, in this case those exploiting a methyl-TROSY effect,¹² which has resulted in applications involving protein complexes in excess of 1 MDa.¹³

The continued success of NMR studies of molecular machines over the past years has led to the study of increasingly complex targets. For example, initial studies by our laboratory focused on the 670 kDa proteasome core particle,¹⁴ that is comprised of 14 copies of α and β-subunits, each with a molecular mass of approximately 25 kDa. The relatively small size of each protomer, albeit within the context of a large complex, meant that resolution was not limiting and that experiments could be performed using samples where methyl groups of Leu and Val were not stereospecifically ¹³CH₃ labeled, with one isopropyl methyl NMR-active in each Leu and Val. As the size of the individual protomers of the complex increases, spectral resolution is challenged as a pair of signals is measured for each isopropyl group. Boisbouvier and coworkers have developed an elegant approach that significantly addresses this limitation by introducing a synthetic route for the production of specifically methyl-labeled acetolactate, subsequently converted into either proR- or proS-¹³CH₃ α-ketoisovalerate in vivo that then produces Leu and Val with ¹³CH₃ labeling confined to a single prochiral methyl¹⁵ (Figure 1A). In addition, to simplifying the resulting NMR spectrum, the sensitivity of cross-peaks is increased two-fold relative to samples prepared with the racemic pro-R/pro-S ¹³CH₃, ¹²CD₃-isovalerate mixture as all of the label is focused on a single position rather than a 50%-50% split. This is an important consideration in applications involving samples that are concentration limited, and, in general in studies of very high-molecular-weight complexes.

Our recent work on the proteostasis protein p9716 provides an example of the utility of the stereospecific biosynthetic labeling scheme of Boisbouvier.¹⁵ Here we focused on a 320 kDa hexameric fragment of p97, with each of the protomers comprising residues 1-480 of the 540 kDa intact protein. A series of p97 disease mutants had been characterized biochemically prior to our study, but little structural information was available that provided a firm understanding of the relation between the mutants and p97 malfunction; indeed X-ray-derived structures of wild-type and mutated proteins were very similar. Figure 1B shows the Leu/Val region of the ¹³C-¹H HMQC spectrum recorded on a sample of highly deuterated, Ile Cδ1-¹³CH₃, proR Leu,Val Cδ,Cα-¹³CH₃, Met Cε-¹³CH₃ p97, highlighting the excellent sensitivity and resolution of the data set, along with cartoon representations of the structures in both ADP and ATP states, as established by X-ray studies. Note that one of the domains (colored purple) changes position significantly between the different nucleotide states. We have been able to show through NMR studies of a series of disease mutants that this domain changes orientation in response to disease severity, establishing a connection between structural-dynamics, and disease that was missing from X-ray analyses.

Molecular machines are ubiquitous in the cell, and they involve moving parts that, in principle, can now be characterized in quantitative detail by solution NMR methods. The development of stereospecific methyl labeling has been an important advance in achieving this goal.

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