Selective Isotopic Unlabeling of Proteins Using Metabolic Precursors: Application to NMR Assignment of Intrinsically Disordered Proteins

Rasia, Rodolfo M.; Brutscher, Bernhard; Plevin, Michael J.

doi:10.1002/cbic.201100678

Cited by 25 publications

(20 citation statements)

References 34 publications

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“…The approach applied here may be viewed as a ''two-dimensional'' combinatorial method, where in the ''sample dimension'' residue types containing a 15 N label are identified from the pattern of presence/absence of cross peaks in HSQC spectra of each sample, and in the ''spectroscopic dimension'' the various combinations of 12 C/ 13 C isotopomeric dipeptide species are edited via the presence/absence of cross peaks in a series of 2D HN(C)-type triple-resonance spectra. In this regard there is a loose analogy to an assignment strategy that combines a precursor-based selective unlabeling protocol with Hadamard-encoded amino acid-type editing to enhance the information that can be gained from a limited set of samples (Rasia et al 2012). …”

Section: Merits Of Combinatorial Selective Labelingmentioning

confidence: 99%

An extended combinatorial 15N, 13Cα, and $$ ^{13} {\text{C}}^{\prime } $$ labeling approach to protein backbone resonance assignment

et al. 2015

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Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in (15)N or (13)C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in (1)H-(15)N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.

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Section: Merits Of Combinatorial Selective Labelingmentioning

confidence: 99%

An extended combinatorial 15N, 13Cα, and $$ ^{13} {\text{C}}^{\prime } $$ labeling approach to protein backbone resonance assignment

et al. 2015

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“…For example, the most powerful application of the methods would be site-specific/desired amino acid selective isotope labeling of proteins, which has been firmly established using the E. coli expression system [22], [23], [24], [25]. Alternative 13 C-labeling of protein is also possible by using particular 13 C-enriched carbon sources, such as [1- 13 C]-glucose [26], [1,3- 13 C 2 ]- or [2- 13 C]-glycerol [27], 13 C-acetate [28], and [1,2- 13 C 2 ]- or [3- 13 C]-pyruvate [29], [30], [31].…”

Section: Protein Sample Preparation For Nmr Measurementsmentioning

confidence: 99%

Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists

Sugiki

Kobayashi

Fujiwara

2017

Computational and Structural Biotechnology Journal

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Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for structural studies of chemical compounds and biomolecules such as DNA and proteins. Since the NMR signal sensitively reflects the chemical environment and the dynamics of a nuclear spin, NMR experiments provide a wealth of structural and dynamic information about the molecule of interest at atomic resolution. In general, structural biology studies using NMR spectroscopy still require a reasonable understanding of the theory behind the technique and experience on how to recorded NMR data. Owing to the remarkable progress in the past decade, we can easily access suitable and popular analytical resources for NMR structure determination of proteins with high accuracy. Here, we describe the practical aspects, workflow and key points of modern NMR techniques used for solution structure determination of proteins. This review should aid NMR specialists aiming to develop new methods that accelerate the structure determination process, and open avenues for non-specialist and life scientists interested in using NMR spectroscopy to solve protein structures.

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“…The experiments together provide the chemical shifts of 15 N, 1 HN, 13 C α , 13 C β , 13 C nuclei of the C-terminal neighbor ('i + 1') of amino acid residue, 'i', which is selectively unlabeled. The experiments, namely, 2D HN(CA)(i + 1), GFT (3,2)D HNCACB(i + 1) and GFT (3,2)D HNCACO(i + 1) are further accelerated by employing non-uniform sampling (NUS) [20][21][22][23][24][25][26]. The methodology is demonstrated on a selectively unalabeled protein sample of ubiquitin.…”

Section: Of 13mentioning

confidence: 99%

“…The measurement time required for acquiring the GFT spectra can be reduced further by using the non-uniform sampling (NUS) approach [24][25][26]. The NUS approach is based on the premise that the conventional method involving linear sampling of interferogram in the inderct dimension requires a lot more number of points although the number of frequencies encoded in the interferogram is much less.…”

Section: Data Acquisition Using Non-uniform Sampling (Nus)mentioning

confidence: 99%

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

2017

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Abstract:In recent years, there has been a growing interest in fast acquisition and analysis of nuclear magnetic resonance (NMR) spectroscopy data for high throughput protein structure determination. Towards this end, rapid data collection techniques and methods to simplify the NMR spectrum such as amino acid selective unlabeling have been proposed recently. Combining these two approaches can speed up further the structure determination process. Based on this idea, we present three new two-dimensional (2D) NMR experiments, which together provide 15 N, 1 HN, 13 C α , 13 C β , 13 C chemical shifts for amino acid residues which are immediate C-terminal neighbors (i + 1) of residues that are selectively unlabeled. These experiments have high sensitivity and can be acquired rapidly using the methodology of G-matrix Fourier transform (GFT) NMR spectroscopy combined with non-uniform sampling (NUS). This is a first study involving the application of fast NMR methods to proteins samples prepared using a specific labeling scheme. Taken together, this opens up new avenues to using the method of selective unlabeling for rapid resonance assignment of proteins.

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Selective Isotopic Unlabeling of Proteins Using Metabolic Precursors: Application to NMR Assignment of Intrinsically Disordered Proteins

Cited by 25 publications

References 34 publications

An extended combinatorial 15N, 13Cα, and $$ ^{13} {\text{C}}^{\prime } $$ labeling approach to protein backbone resonance assignment

An extended combinatorial 15N, 13Cα, and $$ ^{13} {\text{C}}^{\prime } $$ labeling approach to protein backbone resonance assignment

Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

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