Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins

Gans, Pierre; Hamelin, Olivier; Sounier, Rémy; Ayala, Isabel; Durá, M. Asunción; Amero, Carlos; Noirclerc‐Savoye, Marjolaine; Franzetti, Bruno; Plevin, Michael J.; Boisbouvier, Jérôme

doi:10.1002/anie.200905660

Cited by 196 publications

(116 citation statements)

References 29 publications

Supporting

Mentioning

116

Contrasting

Order By: Relevance

“…2A). Maximum isotope incorporation was reached when just ~30 mg/L of precursors were added, an order of magnitude less than the 300 mg/L reported in the original paper (Gans et al, 2010). We tested isotope incorporation into Leu using the strain BL21(AcL-/Val-) with different concentrations of 13 C-monomethyl α-ketoisovalerate and, in contrast to the poor isotope incorporation observed for the Δ ilvE/ Δ avtA strain, we reached maximum incorporation using only 25 mg/L of 13 C-monomethyl α-ketoisovalerate (Fig.…”

Section: Resultsmentioning

confidence: 57%

“…Using the optimized conditions described above, we produced different methyl-labeled samples of the 82 kDa malate synthetase G (MSG), which is one of the largest monomeric proteins with available methyl assignments(Gans et al, 2010; Tugarinov and Kay, 2003). The MSG sequence contains 70 Leu and 46 Val residues.…”

Section: Resultsmentioning

confidence: 99%

“…Owing to their relatively small chemical shift dispersion and overlapping chemical shift ranges, simultaneous labeling of Leu and Val can often result in crowded and difficult to analyze spectra, especially as protein size increases. Simultaneous stereospecific labeling of pro-S or pro-R methyl groups in both Leu and Val via synthesis of specifically labeled acetolactate precursors was shown to alleviate spectral crowding and provides significant sensitivity enhancement in large proteins (Gans et al, 2010). However, there are currently no methods available to separately and stereospecifically label Leu or Val.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

et al. 2016

View full text Add to dashboard Cite

A simple and cost effective method to independently and stereo-specifically incorporate [1H,13C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [1H,13C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [1H,13C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-13C,2,3-2H2,15N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[13CH3]γ2 labeled protein with an optimal isotope incorporation using up to 50% less labeled Thr than the traditional E. coli strain without the need for 2H-glycine to prevent scrambling.

show abstract

Section: Resultsmentioning

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

et al. 2016

View full text Add to dashboard Cite

show abstract

“…The stereo-selective labeling for δ2/γ2 methyl groups was used to distinguish the δ1/γ1 and δ2/γ2 methyl groups in Leu and Val () (Gans et al, 2010). …”

Section: Methodsmentioning

confidence: 99%

Mitosis-specific acetylation tunes Ran effector binding for chromosome segregation

Bao

Liu

et al. 2017

Journal of Molecular Cell Biology

View full text Add to dashboard Cite

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. The Ran GTPase plays a key role in mitotic spindle assembly. However, how the generation of a chemical gradient of Ran-GTP at the spindle is coupled to mitotic post-translational modifications has never been characterized. Here, we solved the complex structure of Ran with the nucleotide release factor Mog1 and delineated a novel mitosis-specific acetylation-regulated Ran–Mog1 interaction during chromosome segregation. Our structure-guided functional analyses revealed that Mog1 competes with RCC1 for Ran binding in a GTP/GDP-dependent manner. Biochemical characterization demonstrated that Mog1-bound Ran prevents RCC1 binding and subsequent GTP loading. Surprisingly, Ran is a bona fide substrate of TIP60, and the acetylation of Lys134 by TIP60 liberates Mog1 from Ran binding during mitosis. Importantly, this acetylation-elicited switch of Ran binding to RCC1 promotes high level of Ran-GTP, which is essential for chromosome alignment. These results establish a previously uncharacterized regulatory mechanism in which TIP60 provides a homeostatic control of Ran-GTP level by tuning Ran effector binding for chromosome segregation in mitosis.

show abstract

“…The different isotope labeling strategies exploit the biosynthetic machinery by supplementing the growth medium with suitably labeled late stage amino acid precursor compounds. Metabolic precursor compounds, such as pyruvate- (Guo et al 2009), α-ketoacid- (Goto et al 1999) or acetolactate-derivatives (Gans et al 2010) have been efficiently applied in cell-based protein expression systems, leading to incorporation of stable isotopes at well-defined positions in the target macromolecules.…”

Section: Specific Isotope-labelling Strategiesmentioning

confidence: 99%

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

Kurzbach

Kontaxis

Coudevylle

et al. 2015

Advances in Experimental Medicine and Biology

View full text Add to dashboard Cite

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational flexibility and thus not amenable to conventional structural biology techniques. Given their inherent structural flexibility NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This chapter will summarize key advances in NMR methodology. Despite the availability of efficient (multi-dimensional) NMR experiments for signal assignment of IDPs it is discussed that NMR of larger and more complex IDPs demands spectral simplification strategies capitalizing on specific isotope-labeling strategies. Prototypical applications of isotope labeling-strategies are described. Since IDP-ligand association and dissociation processes frequently occur on time scales that are amenable to NMR spectroscopy we describe in detail the application of CPMG relaxation dispersion techniques to studies of IDP protein binding. Finally, we demonstrate that the complementary usage of NMR and EPR data provide a more comprehensive picture about the conformational states of IDPs and can be employed to analyze the conformational ensembles of IDPs.

show abstract

Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins

Cited by 196 publications

References 29 publications

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

Mitosis-specific acetylation tunes Ran effector binding for chromosome segregation

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

Contact Info

Product

Resources

About