Category Archives: Structure Determination

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites #DNPNMR

Marin-Montesinos, Ildefonso, David Goyard, Emilie Gillon, Olivier Renaudet, Anne Imberty, Sabine Hediger, and Gaël De Paëpe. “Selective High-Resolution DNP-Enhanced NMR of Biomolecular Binding Sites.” Chemical Science 10, no. 11 (2019): 3366–74.

https://doi.org/10.1039/C8SC05696J.

Locating binding sites in biomolecular assemblies and solving their structures are of the utmost importance to unravel functional aspects of the system and provide experimental data that can be used for structurebased drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR. In this work, we introduce a novel method called Selective Dynamic Nuclear Polarization (Sel-DNP) that allows selective highlighting and identification of residues present in the binding site. This powerful site-directed approach relies on the use of localized paramagnetic relaxation enhancement induced by a ligand-functionalized paramagnetic construct combined with difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites. The identification of residues involved in the binding is performed using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivities. The methodology is demonstrated on the galactophilic lectin LecA, for which we report well-resolved DNP-enhanced spectra with linewidths between 0.5 and 1 ppm, which enable the de novo assignment of the binding interface residues, without using previous knowledge of the binding site location. Since this approach produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the size of the biomolecular system and only requires the production of one protein sample (e.g. 13C,15N-labeled protein).

Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR #DNPNMR

Elkins, Matthew R, Ivan V Sergeyev, and Mei Hong. “Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR.” Journal of the American Chemical Society, 2018, 15437–15449. 

https://doi.org/10.1021/jacs.8b09658

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic 13C enrichment of cholesterol with detection of protein-cholesterol 13C-13C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A 13C-13C double-quantum filter was employed to significantly simplify the 2D 13C-13C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues’ sidechains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.

Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning #DNPNMR

Jaudzems, Kristaps, Andrea Bertarello, Sachin R. Chaudhari, Andrea Pica, Diane Cala-De Paepe, Emeline Barbet-Massin, Andrew J. Pell, et al. “Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning.” Angewandte Chemie 0 (2018).

https://doi.org/10.1002/ange.201801016.

Dynamic nuclear polarization (DNP) is a powerful way to overcome the sensitivity limitation of magic?angle?spinning (MAS) NMR experiments. However, the resolution of the DNP?NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals. High?quality DNP?enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800?MHz) and fast MAS (40?kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar?based experiments. This enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long?range contacts, which are not observed at room temperature, opening new possibilities for structure determination.

The Atomic-Level Structure of Cementitious Calcium Silicate Hydrate

Kumar, A., et al., The Atomic-Level Structure of Cementitious Calcium Silicate Hydrate. The Journal of Physical Chemistry C, 2017. 121(32): p. 17188-17196.

http://dx.doi.org/10.1021/acs.jpcc.7b02439

Efforts to tune the bulk physical properties of concrete are hindered by a lack of knowledge related to the atomic-level structure and growth of calcium silicate hydrate phases, which form about 50–60% by volume of cement paste. Here we describe the first synthesis of compositionally uniform calcium silicate hydrate phases with Ca:Si ratios tunable between 1.0 and 2.0. The calcium silicate hydrate synthesized here does not contain a secondary Ca(OH)2 phase, even in samples with Ca:Si ratios above 1.6, which is unprecedented for synthetic calcium silicate hydrate systems. We then solve the atomic-level three-dimensional structure of these materials using dynamic nuclear polarization enhanced 1H and 29Si nuclear magnetic resonance experiments in combination with atomistic simulations and density functional theory chemical shift calculations. We discover that bridging interlayer calcium ions are the defining structural characteristic of single-phase cementitious calcium silicate hydrate, inducing the strong hydrogen bonding that is responsible for stabilizing the structure at high Ca:Si ratios.

Measuring Nano- to Microstructures from Relayed Dynamic Nuclear Polarization NMR #DNPNMR

Pinon, A.C., et al., Measuring Nano- to Microstructures from Relayed Dynamic Nuclear Polarization NMR. The Journal of Physical Chemistry C, 2017. 121(29): p. 15993-16005.

http://dx.doi.org/10.1021/acs.jpcc.7b04438

We show how dynamic nuclear polarization (DNP) NMR can be used in combination with models for polarization dynamics to determine the domain sizes in complex materials. By selectively doping a source component with radicals and leaving the target undoped, we can measure experimental polarization buildup curves which can be compared with simulations based on heterogeneous distributions of polarization within the sample. The variation of the integrated DNP enhancement as a function of the polarization time is found to be characteristic of the geometry. We demonstrate the method experimentally on four different systems where we successfully determine domain sizes between 200 and 20 000 nm, specifically in powdered histidine hydrochloride monohydrate, pore lengths of mesoporous silica materials, and two domain sizes in two-component polymer film coatings. Additionally, we find that even in the apparently homogeneous frozen solutions used as polarization sources in most DNP experiments, polarization is relayed from protons near the radicals to the bulk of the solution by spin diffusion, which explains the experimentally observed buildup times in these samples.

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR #DNPNMR

Ni, Q.Z., et al., Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR. The Journal of Physical Chemistry B, 2017. 121(19): p. 4997-5006.

http://dx.doi.org/10.1021/acs.jpcb.7b02066

In DNP MAS NMR experiments at ∼80–110 K, the structurally important −13CH3 and −15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78–300 K. In APG, the three-site hopping of the Ala-Cβ peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C’s and 15N’s. Similarly, the 15N signal from Ala-NH3+ disappears at ∼173 K, concurrent with the attenuation in CP experiments of other 15N’s as well as 13C’s. In bR and PI3-SH3, the methyl groups are attenuated at ∼95 K, while all other 13C’s remain unaffected. However, both systems exhibit substantial losses of intensity at ∼243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy #DNPNMR

Nagaraj, M., et al., Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy. Chembiochem, 2016. 17(14): p. 1308-11.

https://www.ncbi.nlm.nih.gov/pubmed/27147408

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.

Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization #DNPNMR

Viennet, T., et al., Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization. Angew Chem Int Ed Engl, 2016. 55(36): p. 10746-50.Viennet, T., et al., Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization. Angew Chem Int Ed Engl, 2016. 55(36): p. 10746-50.

https://www.ncbi.nlm.nih.gov/pubmed/27351143

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments.

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR #DNPNMR

Lange, S., et al., Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR. Sci Adv, 2016. 2(8): p. e1600379.

https://www.ncbi.nlm.nih.gov/pubmed/27551685

Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.

A New Tool for NMR Crystallography: Complete (13)C/(15)N Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid-State NMR

Marker, K., et al., A New Tool for NMR Crystallography: Complete (13)C/(15)N Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid-State NMR. J Am Chem Soc, 2015. 137(43): p. 13796-9.

http://www.ncbi.nlm.nih.gov/pubmed/26485326

NMR crystallography of organic molecules at natural isotopic abundance (NA) strongly relies on the comparison of assigned experimental and computed NMR chemical shifts. However, a broad applicability of this approach is often hampered by the still limited (1)H resolution and/or difficulties in assigning (13)C and (15)N resonances without the use of structure-based chemical shift calculations. As shown here, such difficulties can be overcome by (13)C-(13)C and for the first time (15)N-(13)C correlation experiments, recorded with the help of dynamic nuclear polarization. We present the complete de novo (13)C and (15)N resonance assignment at NA of a self-assembled 2′-deoxyguanosine derivative presenting two different molecules in the asymmetric crystallographic unit cell. This de novo assignment method is exclusively based on aforementioned correlation spectra and is an important addition to the NMR crystallography approach, rendering firstly (1)H assignment straightforward, and being secondly a prerequisite for distance measurements with solid-state NMR.

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