Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris (original) (raw)
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Comprehensive and Cost-Effective NMR Spectroscopy of Methyl Groups in Large Proteins
Journal of the American Chemical Society, 2010
An NMR approach is described which yields the methyl resonance assignments of alanine, threonine, valine, leucine, and isoleucine residues in proteins with high sensitivity and excellent resolution. The method relies on protein samples produced by bacterial expression using U-[ 1 H, 13 C]-D-glucose and ∼100% D 2 O, which is cost-effective and ensures the isotopic enrichment of all possible methyl groups. Magnetization transfer throughout the methyl-containing side chains is possible with this labeling scheme due to the high level of deuteration along the amino acid side chain, coupled with the selection of the favorable 13 CHD 2 methyl isotopomer for detection. In an application to the 34 kDa periplasmic binding protein FepB, 164 out of 195 methyl groups (85%) were assigned sequence-specifically and stereospecifically. This percentage increases to 91% when taking into account that not all backbone assignments are available for this system. The remaining unassigned methyl groups belong to six leucine residues, caused by low crosspeak intensities, and four alanine residues due to degeneracy of the 13 C α / 13 C β frequencies. Our results demonstrate that NMR spectroscopic investigations of protein structure, dynamics, and interactions can be extended to include all methylcontaining amino acids also for larger proteins.
Automatic structure-based NMR methyl resonance assignment in large proteins
2019
Isotope-labeled methyl groups provide NMR probes that can be observed in very large, otherwise deuterated systems and enable investigations of protein structure, dynamics and mechanisms. However, the assignment of resonances to specific methyls in the protein is expensive and time-consuming, which limits the use of methyl-based NMR for large proteins. To resolve this bottleneck, methyl assignment methods have been developed. However, these remain limited regarding complete automation, computational feasibility, and/or the extent and accuracy of the assignments. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA was applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 674 had manually determined 1H/13C reference assignments and 614 showed cross peaks in the available NOESY peak lists. MethylFLYA confi...
2001
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1 H/ 15 N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15 N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1 H/ 15 N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15 N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1 H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
2001
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1 H/ 15 N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15 N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1 H/ 15 N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15 N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1 H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
Assigning methyl resonances for protein solution-state NMR studies
Methods, 2018
Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (> 50 kDa) proteins. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.
Journal of Biomolecular NMR, 2013
The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new "redox" approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-13 C] acetate does not label carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one-and two-bond dipolar couplings provides narrow signals and long-range, intra-and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-13 C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra.
Labeling strategies for 13C-detected aligned-sample solid-state NMR of proteins
Journal of Magnetic Resonance, 2009
13 C-detected solid-state NMR experiments have substantially higher sensitivity than the corresponding 15 N-detected experiments on stationary, aligned samples of isotopically labeled proteins. Several methods for tailoring the isotopic labeling are described that result in spatially isolated 13 C sites so that dipole-dipole couplings among the 13 C are minimized, thus eliminating the need for homonuclear 13 C-13 C decoupling in either indirect or direct dimensions of one-or multidimensional NMR experiments that employ 13 C detection. The optimal percentage for random fractional 13 C labeling is between 25% and 35%. Specifically labeled glycerol and glucose can be used at the carbon sources to tailor the isotopic labeling, and the choice depends on the resonances of interest for a particular study. For investigations of the protein backbone, growth of the bacteria on 2-13 C-glucose containing media was found to be most effective.
Asymmetric Methyl Group Labeling as a Probe of Membrane Protein Homo-oligomers by NMR Spectroscopy
Journal of the American Chemical Society, 2008
Homo-and hetero-oligomeric membrane protein interactions are central to many cellular events, including signal transduction, post-translational modification, and regulation of receptors, channels, and several other integral membrane proteins. 1 Since the majority of membrane proteins are helical, these interactions often involve helix-helix packing, with interdigitation of hydrophobic side chains (Leu, Val, Ile, Ala) to form strong van der Waals interactions, that is, the "knobs-into-holes" model proposed by Crick. 2 Yet probing these hydrophobic binding surfaces remains a formidable challenge for structural techniques. NMR is a powerful tool for investigating dynamic and functional interfaces of soluble proteins. 3,4 Chemical shift mapping, saturation transfer, paramagnetic relaxation enhancement, and cross-relaxation (nuclear Overhauser effect, NOE) are among the NMR methods currently used. By probing the through-space cross-relaxation of dipolarly coupled nuclei, NOE is considered a direct probe of molecular interfaces. Intermolecular NOEs are detected using isotope-filtered experiments 5 in differentially labeled proteins. 6,7 While applied with success to several medium size complexes thanks to the implementation of transverse relaxation optimized spectroscopy (TROSY) and partial protein deuteration, these methods are very insensitive. Jasanoff also introduced an asymmetric labeling method, where the detection of intermolecular NOEs in homo-oligomers relies on the comparison of two 13 C-edited NOESY spectra. 8 As with the isotope-filtered experiments, this approach is severely limited by spectral overlapping of the aliphatic groups, the need to acquire two NOESY spectra, and the ability to detect only short-range distances (~6 Å). Recently Kay and co-workers introduced the selective methyl labeling techniques in conjunction with methyl-NOESY pulse sequences, pushing both sensitivity and resolution of NMR up to molecular weights of ~1 MDa. 9-11 The isotopic labeling of [U-2 H, 13 C, 15 N] with 13 CH 3 methyl labels at Ile, Val, and Leu allowed for the identification of NOEs between methyl groups to give tertiary restraints in soluble proteins with hydrophobic cores at highresolution. However, for hetero-oligomeric protein interactions, it is difficult to resolve intermolecular NOEs on the basis of this labeling scheme because of severe overlap of the resonances, 12 and in the case of symmetric homo-oligomers this problem is exacerbated by the magnetic equivalence of the methyl groups. In this Communication, we present an asymmetric isotopic labeling strategy for probing unambiguously membrane protein binding interfaces in homo-oligomers. We show that by
Optimal isotope labelling for NMR protein structure determinations
Nature, 2006
Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.