Amino Acid Selective Labeling and Unlabeling for Protein Resonance Assignments

Jaipuria, Garima; Krishnarjuna, Bankala; Mondal, Somnath; Dubey, A.; Atreya, Hanudatta S.

doi:10.1007/978-94-007-4954-2_6

Cited by 29 publications

(24 citation statements)

References 62 publications

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“…As described above, conventional CSL schemes generally uses two SI-labeling levels, enabling that 1 bit information is contained in each labeled sample (Parker et al 2004;Shi et al 2004;Trbovic et al 2005;Staunton et al 2006;Wu et al 2006;Maslennikov et al 2010;Sobhanifar et al 2010;Hefke et al 2011;Krishnarjuna et al 2011;Jaipuria et al 2012; Maslennikov and Choe 2013). In the previously mentioned triple selective CSL approach (Löhr et al 2012), three SIlabeling types can be discriminated for both residues i and i -1: unlabeled, 15 N-labeled, or 2-13 C/ 15 N-labeled for the residue i and unlabeled, 1-13 C-labeled, or 1,2-13 C-labeled for the residue i -1, respectively, being considered that 1 trit (ternary digit) information is contained in each sample.…”

Section: Discussionmentioning

confidence: 99%

“…However, for the discrimination of all amino-acids, these simple AASIL schemes require a large number of samples, which are typically the same as the number of amino acids (19 for nitrogen or 20 for carbon). For this reason, various combinatorial selective labeling (CSL) schemes (Parker et al 2004;Shi et al 2004;Trbovic et al 2005;Staunton et al 2006;Wu et al 2006;Maslennikov et al 2010;Sobhanifar et al 2010;Hefke et al 2011;Krishnarjuna et al 2011;Jaipuria et al 2012;Löhr et al 2012; Maslennikov and Choe 2013) were developed to reduce required number of samples, by representing amino acids as combination of SI labeled samples rather than simply assigning one amino acid to one SI labeled sample. For example, a CSL scheme developed by Parker et al (2004), which is based on the dual selective approach, can discriminate 16 amino-acids with one uniformly 13 C and 15 N-labeled reference and four selectively (100 or 0 % for 13 C and 100 or 50 % for 15 N, respectively) labeled samples.…”

Section: Introductionmentioning

confidence: 99%

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Stable isotope labeling strategy based on coding theory

et al. 2015

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We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as ''encoding and decoding'' processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Stable isotope labeling strategy based on coding theory

et al. 2015

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“…Aiming at full backbone assignments, separate samples with different pairs of amino acids would have to be prepared, and data to be collected on, to an extent of the same order of magnitude as the number of residues present in the protein, which is clearly unfeasible. To this end, recently reviewed combinatorial methods (Jaipuria et al 2012) using multiple mixtures, each containing different subsets of labeled amino acids rather than a single of each labeling type, are much more efficient (Parker et al 2004;Shi et al 2004;Shortle 1994;Staunton et al 2006;Trbovic et al 2005;Wu et al 2006).…”

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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“…Relaxation experiments are not the only case when specific isotopic labeling is necessary, other examples are methyl group labeling for very large systems [67], segmental labeling for multi domain protein studies [68] and amino acid selective labeling and unlabeling for protein assignments [69].…”

Section: Specific Isotopic Labelingmentioning

confidence: 99%

Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain

Ahlner¹

2015

Linköping Studies in Science and Technology. Dissertations

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Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins.To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the software also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multiv dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG relaxation dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals.To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of E...

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Amino Acid Selective Labeling and Unlabeling for Protein Resonance Assignments

Cited by 29 publications

References 62 publications

Stable isotope labeling strategy based on coding theory

Stable isotope labeling strategy based on coding theory

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

Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain

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