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Conformational dynamics and alignment properties of loop lanthanide-binding-tags (LBTs) studied in interleukin-1β
May 23, 2017 - 4:44 AM - by nmrlearner
nmrlearner's Avatar Conformational dynamics and alignment properties of loop lanthanide-binding-tags (LBTs) studied in interleukin-1β

Abstract

Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1β (Barthelmes et al. in J Am Chem Soc 133:808??819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1β constructs by measuring 15N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-?c-timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-?c dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.



Source: Journal of Biomolecular NMR
0 Replies | 1 Views
Molecular dynamics-based selectivity for Fast-Field-Cycling relaxometry by Overhauser and solid effect dynamic nuclear polarization #DNPNMR
May 23, 2017 - 4:44 AM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

Molecular dynamics-based selectivity for Fast-Field-Cycling relaxometry by Overhauser and solid effect dynamic nuclear polarization #DNPNMR

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Neudert, O., C. Mattea, and S. Stapf, Molecular dynamics-based selectivity for Fast-Field-Cycling relaxometry by Overhauser and solid effect dynamic nuclear polarization. J. Magn. Reson., 2017. 276: p. 113-121.


http://www.sciencedirect.com/science...90780717300204


In the last decade nuclear spin hyperpolarization methods, especially Dynamic Nuclear Polarization (DNP), have provided unprecedented possibilities for various NMR techniques by increasing the sensitivity by several orders of magnitude. Recently, in-situ DNP-enhanced Fast Field Cycling (FFC) relaxometry was shown to provide appreciable NMR signal enhancements in liquids and viscous systems. In this work, a measurement protocol for DNP-enhanced NMR studies is introduced which enables the selective detection of nuclear spin hyperpolarized by either Overhauser effect or solid effect DNP. Based on field-cycled DNP and relaxation studies it is shown that these methods allow for the independent measurement of polymer and solvent nuclear spins in a concentrated solution of high molecular weight polybutadiene in benzene doped with ?,?-bisdiphenylene-?-phenylallyl radical. Appreciable NMR signal enhancements of about 10-fold were obtained for both constituents.... [Read More]
0 Replies | 1 Views
[NMR paper] NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris.
May 22, 2017 - 10:55 AM - by nmrlearner
nmrlearner's Avatar NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris.

NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris.

Biochim Biophys Acta. 2017 May 17;:

Authors: Khairutdinov BI, Ermakova EA, Yusypovych YM, Bessolicina EK, Tarasova NB, Toporkova YY, Kovaleva V, Zuev YF, Nesmelova IV

Abstract
Plants have developed a complex defense response system against pests and pathogens. Defensins, produced by plants as part of their innate immune response, form the family of small, basic, cysteine-rich proteins with activity primarily directed against fungal pathogens. In addition, plant defensins can show antibacterial activity and protease and insect amylase inhibitory activities. However, in gymnosperms, only antifungal activity of defensins has been described thus far. Here, we report antibacterial and insect ?-amylase inhibition activities for defensin PsDef1 from P. sylvestris, the first defensin from gymnosperms with a broad range of biological activities described. We also report the solution NMR structure of PsDef1 and its dynamics properties assessed by a combination of experimental NMR and computational techniques. Collectively, our data provide an insight into structure, dynamics, and functional properties of PsDef1 that could be common between defensins from this taxonomic group.


PMID: 28528214 [PubMed - as supplied by publisher]



... [Read More]
0 Replies | 5 Views
[NMR paper] Double-Caging Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and in situ Quantification
May 19, 2017 - 4:40 PM - by nmrlearner
nmrlearner's Avatar Double-Caging Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and in situ Quantification


We report on in situ fluorescent quantification of the conjugation efficiency between azide-terminated synthetic polymers/ imaging probes and thiol-functionalized antibodies/proteins/peptides, by utilizing a doubly caged profluorescent and heterodifunctional core molecule (C1) as the self-sorting bridging unit. Orthogonal dual 'click' coupling of C1 with azide- and thiol-functionalized precursors leads to highly fluorescent bioconjugates, whereas single click products of C1 remain essentially nonfluorescent. This 'AND' logic gate-type fluorogenic feature also enables further integration with FRET processes. For the construction of antibody-probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual 'click' coupling process with C1 can be conveniently monitored via emission turn-on of C1, whereas prominent changes in FRET ratios occur for antibody-probe conjugates when triggered by specific tumor-associated enzymes.

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0 Replies | 13 Views
T1 - Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags #DNPNMR
May 19, 2017 - 4:40 PM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

T1 - Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags #DNPNMR

Rivkah Rogawski, Ivan V. Sergeyev, Yongjun Li, M. Francesca Ottaviani, Virginia Cornish, and Ann E. McDermott The Journal of Physical Chemistry B 2017 121 (6), 1169-1175


http://dx.doi.org/10.1021/acs.jpcb.6b09021


Dynamic nuclear polarization is an emerging technique for sensitizing solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable biradicals, the polarization source for the cross effect mechanism, are typically codissolved at millimolar concentrations with proteins of interest. Here we describe the high-affinity biradical tag TMP-T, created by covalently linking trimethoprim, a nanomolar affinity ligand of dihydrofolate reductase (DHFR), to the biradical polarizing agent TOTAPOL. With TMP-T bound to DHFR, large enhancements of the protein spectrum are observed, comparable to when TOTAPOL is codissolved with the protein. In contrast to TOTAPOL, the tight binding TMP-T can be added stoichiometrically at radical concentrations orders of magnitude lower than in previously described preparations. Benefits of the reduced radical concentration include reduced spectral bleaching, reduced chemical perturbation of the sample, and the ability to selectively enhance signals for the protein of interest.


Go to ... [Read More]
0 Replies | 13 Views
[NMR] Postdoctoral position in structural biology of membrane remodeling
May 19, 2017 - 4:01 AM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

[NMR] Postdoctoral position in structural biology of membrane remodeling





POSTDOCTORAL POSITION AVAILABLE
STRUCTURAL BIOLOGY OF MEMBRANE REMODELING


A NIH-funded postdoctoral position is available immediately in the Ramachandran Lab at Case Western Reserve University (CWRU) to study the structural aspects of protein-mediated membrane remodeling during endocytic and mitochondrial membrane fission. This position involves extensive collaboration with the lab of Patrick van der Wel at the University of Pittsburgh. The position requires a Ph.D. in biochemistry or biophysics with a focus on structural biology or membrane biophysics (NMR or ssNMR, preferably). This position will provide an excellent opportunity to learn and apply a wide array of structural and biophysical techniques to explore protein function on a model membrane surface. The Ramachandran laboratory also employs a host of cutting-edge spectroscopic approaches including FRET, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging (FLIM) to explore protein-protein and protein-membrane interactions in membrane remodeling and fission, both in vitro and in vivo. The Ramachandran and Van der Wel labs and the facilities at CWRU and University of Pittsburgh are equipped with state-of-the-art instrumentation for both biophysical techniques and structural biology, as well as for protein purification, characterization and membrane reconstitution.


Requirements: Applicants must be highly motivated and must have demonstrated experience (i.e. relevant publications) in protein biochemistry and structural biology. The candidate should have a strong conceptual and... [Read More]
0 Replies | 11 Views
[NMR] Position as Research Engineer / Lab Manager for the magnetic resonance lab @ IPF Dresden
May 19, 2017 - 4:01 AM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

[NMR] Position as Research Engineer / Lab Manager for the magnetic resonance lab @ IPF Dresden



Position as Research Engineer / Lab Manager for the magnetic resonance lab @ IPF Dresden


At the Institute of Physical Chemistry and Polymer Physics, Department Polyelectrolytes and Dispersions, at the


Leibniz-Institut fr Polymerforschung is an opening for a Research Engineer / Lab Manager for the magnetic resonance laboratory.


We are looking for a motivated, skillful person, who is willing to learn about novel technolgies and is closely collaborating with the PI's. The research engineer will be responsible for ensuring the operation of the laboratory and the various spectrometers (high-field NMR, low-field NMR and X-band EPR), perform routine experiments and sample preparation and support in simulations. Ensuring the smooth operation of the lab includes trouble shooting and modification of experimental setups. Skills in electronics and computing are desirable.


Qualification: MSc/Diploma in Physical Technology or equivalent
Duration: initially for two years


The Leibniz-Institut fr Polymerforschung Dresden e.V. (IPF) is one of the largest polymer research facilities in Germany. As an institute of the Leibniz Association, the IPF is committed to carrying out application-oriented basic research. The focus of activities at the IPF is directed toward the advancement of basic scientific knowledge for the development of functional polymer materials and polymer materials with new or improved characteristics. Leading scientists of the IPF are at the same time appointed professors at the Technische... [Read More]
0 Replies | 12 Views
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