Journal Cover Journal of Biomolecular NMR
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   ISSN (Print) 1573-5001 - ISSN (Online) 0925-2738
   Published by Springer-Verlag Homepage  [2350 journals]
  • 13 C APSY-NMR for sequential assignment of intrinsically disordered
    • Authors: Maria Grazia Murrali; Marco Schiavina; Valerio Sainati; Wolfgang Bermel; Roberta Pierattelli; Isabella C. Felli
      Pages: 167 - 175
      Abstract: The increasingly recognized biological relevance of intrinsically disordered proteins requires a continuous expansion of the tools for their characterization via NMR spectroscopy, the only technique so far able to provide atomic-resolution information on these highly mobile macromolecules. Here we present the implementation of projection spectroscopy in 13C-direct detected NMR experiments to achieve the sequence specific assignment of IDPs. The approach was used to obtain the complete backbone assignment at high temperature of α-synuclein, a paradigmatic intrinsically disordered protein.
      PubDate: 2018-03-01
      DOI: 10.1007/s10858-018-0167-4
      Issue No: Vol. 70, No. 3 (2018)
  • Detection of side-chain proton resonances of fully protonated biosolids in
           nano-litre volumes by magic angle spinning solid-state NMR
    • Authors: James Tolchard; Manoj Kumar Pandey; Mélanie Berbon; Abdelmajid Noubhani; Sven J Saupe; Yusuke Nishiyama; Birgit Habenstein; Antoine Loquet
      Pages: 177 - 185
      Abstract: We present a new solid-state NMR proton-detected three-dimensional experiment dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C–13C transfer to establish efficient (H)CCH correlations detected on side chain protons. Our approach is demonstrated on the HET-s prion domain in its functional amyloid fibrillar form, fully protonated, with a sample amount of less than 500 µg using a MAS frequency of 70 kHz. The majority of aliphatic and aromatic side chain protons (70%) are observable, in addition to Hα resonances, in a single experiment providing a complementary approach to the established proton-detected amide-based multidimensional solid-state NMR experiments for the study and resonance assignment of biosolid samples, in particular for aromatic side chain resonances.
      PubDate: 2018-03-01
      DOI: 10.1007/s10858-018-0168-3
      Issue No: Vol. 70, No. 3 (2018)
  • Measuring the signs of the methyl 1 H chemical shift differences between
           major and ‘invisible’ minor protein conformational states using methyl
           1 H multi-quantum spectroscopy
    • Authors: Anusha B. Gopalan; Pramodh Vallurupalli
      Pages: 187 - 202
      Abstract: Carr–Purcell–Meiboom–Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and ‘invisible’ minor states. The exchange rate(s) ( \( k_{{{\text{ex}}}} \) ), population(s) of the minor state(s) and the absolute value of the chemical shift difference \( {\Delta \varpi } \) (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of \({\Delta \varpi }\) that is required to reconstruct the spectrum of the ‘invisible’ minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl \( ^{1} {\text{H}} \) CPMG experiment (Yuwen in Angew Chem 55:11490–11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of \({\Delta \varpi }\) is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~ 18 kDa cavity mutant of T4 Lysozyme ( \( k_{{{\text{ex}}}} \sim\,3500{\text{ s}}^{{ - 1}} \) ) and (2) the \(\sim\,4.7\)  kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus ( \(k_{ex} \sim\,13,000\hbox { s}^{-1}\) ).
      PubDate: 2018-03-01
      DOI: 10.1007/s10858-018-0171-8
      Issue No: Vol. 70, No. 3 (2018)
  • Correction to: Characterization of fibril dynamics on three timescales by
           solid-state NMR
    • Authors: Albert A. Smith; Emilie Testori; Riccardo Cadalbert; Beat H. Meier; Matthias Ernst
      Pages: 203 - 203
      Abstract: In our recent publication (Smith et al., J Biomol NMR 65:171–191, 2016) on the dynamics of HET-s(218–289), we reported on page 176, that calculation of solid-state NMR R1ρ rate constants using analytical equations based on Redfield theory (Kurbanov et al., J Chem Phys 135:184104:184101–184109, 2011) failed when the correlation time of motion becomes too long.
      PubDate: 2018-03-01
      DOI: 10.1007/s10858-018-0170-9
      Issue No: Vol. 70, No. 3 (2018)
  • Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid
           state NMR
    • Authors: Sebanti Gupta; Robert Tycko
      Pages: 103 - 114
      Abstract: Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716–730, 2010; J Mol Biol 426:1109–1127, 2014; J Biol Chem 291:13098–13112, 2016; J Am Chem Soc 138:8538–8546, 2016; J Am Chem Soc 138:12029–12032, 2016; J Am Chem Soc 134:6455–6466, 2012; J Am Chem Soc 132:1976–1987, 2010; J Am Chem Soc 135:17793–17803, 2013; Proc Natl Acad Sci USA 112:14617–14622, 2015; J Am Chem Soc 138:14066–14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly 15N,13C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.
      PubDate: 2018-02-01
      DOI: 10.1007/s10858-017-0162-1
      Issue No: Vol. 70, No. 2 (2018)
  • Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human
           blood group A and B glycosyltransferases using lanthanide-induced
           pseudocontact shifts and methyl–methyl NOESY
    • Authors: Friedemann Flügge; Thomas Peters
      Abstract: Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl–methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.
      PubDate: 2018-04-26
      DOI: 10.1007/s10858-018-0183-4
  • Production of isotope-labeled proteins in insect cells for NMR
    • Authors: Bastian Franke; Christian Opitz; Shin Isogai; Anne Grahl; Leonildo Delgado; Alvar D. Gossert; Stephan Grzesiek
      Abstract: Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host.
      PubDate: 2018-04-23
      DOI: 10.1007/s10858-018-0172-7
  • Atomic structures of excited state A–T Hoogsteen base pairs in duplex
           DNA by combining NMR relaxation dispersion, mutagenesis, and chemical
           shift calculations
    • Authors: Honglue Shi; Mary C. Clay; Atul Rangadurai; Bharathwaj Sathyamoorthy; David A. Case; Hashim M. Al-Hashimi
      Abstract: NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson–Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A–T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3′ and C4′ spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A–T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A–T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.
      PubDate: 2018-04-19
      DOI: 10.1007/s10858-018-0177-2
  • TROSY pulse sequence for simultaneous measurement of the 15 N R 1 and { 1
           H}– 15 N NOE in deuterated proteins
    • Authors: Paul A. O’Brien; Arthur G. Palmer
      Abstract: A TROSY-based NMR experiment is described for simultaneous measurement of the 15N longitudinal relaxation rate constant R1 and the {1H}–15N nuclear Overhauser enhancement. The experiment is based on the observation that the TROSY mixing pulse sequence element symmetrically exchanges 1H and 15N magnetizations. The accuracy of the proposed technique is validated by comparison to independent measurements of both relaxation parameters for the protein ubiquitin. The simultaneous experiment is approximately 20–33% shorter than conventional sequential measurements.
      PubDate: 2018-04-16
      DOI: 10.1007/s10858-018-0181-6
  • Isotope labeling for studying RNA by solid-state NMR spectroscopy
    • Authors: Alexander Marchanka; Christoph Kreutz; Teresa Carlomagno
      Abstract: Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.
      PubDate: 2018-04-12
      DOI: 10.1007/s10858-018-0180-7
  • Fast evaluation of protein dynamics from deficient 15 N relaxation data
    • Authors: Łukasz Jaremko; Mariusz Jaremko; Andrzej Ejchart; Michał Nowakowski
      Abstract: Simple and convenient method of protein dynamics evaluation from the insufficient experimental 15N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse 15N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S2 and R ex , can be elucidated. The generalized order parameter, S2, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R ex , identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.
      PubDate: 2018-03-28
      DOI: 10.1007/s10858-018-0176-3
  • Trimethylsilyl tag for probing protein–ligand interactions by NMR
    • Authors: Walter Becker; Luke A. Adams; Bim Graham; Gabriel E. Wagner; Klaus Zangger; Gottfried Otting; Christoph Nitsche
      Abstract: Protein–ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B–NS3 protease and the human prolyl isomerase FK506 binding protein.
      PubDate: 2018-03-21
      DOI: 10.1007/s10858-018-0173-6
  • Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein
    • Authors: Kornelia M. Mikula; Luisa Krumwiede; Andreas Plückthun; Hideo Iwaï
      Abstract: Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.
      PubDate: 2018-03-13
      DOI: 10.1007/s10858-018-0175-4
  • Deuteration and selective labeling of alanine methyl groups of β 2
           -adrenergic receptor expressed in a baculovirus-insect cell expression
    • Authors: Yutaka Kofuku; Tomoki Yokomizo; Shunsuke Imai; Yutaro Shiraishi; Mei Natsume; Hiroaki Itoh; Masayuki Inoue; Kunio Nakata; Shunsuke Igarashi; Hideyuki Yamaguchi; Toshimi Mizukoshi; Ei-ichiro Suzuki; Takumi Ueda; Ichio Shimada
      Abstract: G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.
      PubDate: 2018-03-08
      DOI: 10.1007/s10858-018-0174-5
  • Stable isotope labeling approaches for NMR characterization of
           glycoproteins using eukaryotic expression systems
    • Authors: Saeko Yanaka; Hirokazu Yagi; Rina Yogo; Maho Yagi-Utsumi; Koichi Kato
      Abstract: Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinct N-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrate–protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150 kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed.
      PubDate: 2018-02-28
      DOI: 10.1007/s10858-018-0169-2
  • POTENCI: prediction of temperature, neighbor and pH-corrected chemical
           shifts for intrinsically disordered proteins
    • Authors: Jakob Toudahl Nielsen; Frans A. A. Mulder
      Abstract: Chemical shifts contain important site-specific information on the structure and dynamics of proteins. Deviations from statistical average values, known as random coil chemical shifts (RCCSs), are extensively used to infer these relationships. Unfortunately, the use of imprecise reference RCCSs leads to biased inference and obstructs the detection of subtle structural features. Here we present a new method, POTENCI, for the prediction of RCCSs that outperforms the currently most authoritative methods. POTENCI is parametrized using a large curated database of chemical shifts for protein segments with validated disorder; It takes pH and temperature explicitly into account, and includes sequence-dependent nearest and next-nearest neighbor corrections as well as second-order corrections. RCCS predictions with POTENCI show root-mean-square values that are lower by 25–78%, with the largest improvements observed for 1Hα and 13C′. It is demonstrated how POTENCI can be applied to analyze subtle deviations from RCCSs to detect small populations of residual structure in intrinsically disorder proteins that were not discernible before. POTENCI source code is available for download, or can be deployed from the URL
      PubDate: 2018-02-05
      DOI: 10.1007/s10858-018-0166-5
  • Simultaneous detection of intra- and inter-molecular paramagnetic
           relaxation enhancements in protein complexes
    • Authors: Cristina Olivieri; Manu Veliparambil Subrahmanian; Youlin Xia; Jonggul Kim; Fernando Porcelli; Gianluigi Veglia
      Abstract: Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein–protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (15N or 13C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein–protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (1HN-Γ2-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric protein–protein complexes.
      PubDate: 2018-02-02
      DOI: 10.1007/s10858-018-0165-6
  • (3, 2)D 1 H, 13 C BIRD r,X -HSQC-TOCSY for NMR structure elucidation of
           mixtures: application to complex carbohydrates
    • Authors: Natalia Brodaczewska; Zuzana Košťálová; Dušan Uhrín
      Abstract: Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of 1H chemical shifts; larger dispersion of 13C chemical shifts is often used to reduce this overlap, while still providing the proton–proton correlation information e.g. in the form of a 2D 1H, 13C HSQC-TOCSY experiment. For this methodology to work, 13C chemical shift must be resolved. In case of 13C chemical shifts overlap, 1H chemical shifts can be used to achieve the desired resolution. The proposed (3, 2)D 1H, 13C BIRDr,X-HSQC-TOCSY experiment achieves this while preserving singlet character of cross peaks in the F1 dimension. The required high-resolution in the 13C dimension is thus retained, while the cross peak overlap occurring in a regular HSQC-TOCSY experiment is eliminated. The method is illustrated on the analysis of a complex carbohydrate mixture obtained by depolymerisation of a fucosylated chondroitin sulfate isolated from the body wall of the sea cucumber Holothuria forskali.
      PubDate: 2018-01-11
      DOI: 10.1007/s10858-018-0163-8
  • Whole cell solid-state NMR study of Chlamydomonas reinhardtii microalgae
    • Authors: Alexandre A. Arnold; Jean-Philippe Bourgouin; Bertrand Genard; Dror E. Warschawski; Réjean Tremblay; Isabelle Marcotte
      Abstract: In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically 13C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically 13C labelled micro-organisms.
      PubDate: 2018-01-11
      DOI: 10.1007/s10858-018-0164-7
  • Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in
           situ protein NMR analysis
    • Authors: Jia-Liang Chen; Yu Zhao; Yan-Jun Gong; Bin-Bin Pan; Xiao Wang; Xun-Cheng Su
      Abstract: Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein–ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.
      PubDate: 2017-12-09
      DOI: 10.1007/s10858-017-0160-3
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