iNEXT-Discovery: Scientific publications




Publications from iNEXT-Discovery acknowledging iNEXT-Discovery are appearing as Open Access





March 30, 2021 

3D Heteronuclear Magnetization Transfers for the Establishment of Secondary Structures in SARS-CoV-2-Derived RNAs

A heteronuclear-resolved version of NOESY experiments is presented, in which magnetization transfers between the aqueous solvent and the nucleic acid protons are controlled by selecting specific chemical shift combinations of a coupled 1H-15N spin pair. This leads to a pseudo-3D HSQC-NOESY with cross-peaks enhanced by ~2-5 times compared with conventional 2D NOESY. The enhanced signal sensitivity as well as access to both 15N-1H and 1H-1H NOESY dimensions can greatly facilitate RNA assignments and secondary structure determinations.
Kim J, Novakovic M, Jayanthi S, Lupulescu A, Kupce E, Grün JT, Mertinkus K, Oxenfarth A, Richter C, Schnieders R, Wirmer-Bartoschek J, Schwalbe H, Frydman L.
doi: 10.1021/jacs.1c01914
March 26, 2021 

1H, 13C, and 15N backbone chemical‑shift assignments of SARS‑CoV‑2 non‑structural protein 1 (leader protein)

The near complete backbone chemical shift assignment of full-length SARS-CoV-2 Nsp1 is presented, revealing its domain organization, secondary structure and backbone dynamics. The results will be of value to further MNR-based investigations of the biochemical and physiological functions of Nsp1.
Ying Wang Y, Kirkpatrick J, zur Lage S, Korn SM, Neißner K, Schwalbe H, Schlundt A, Carlomagno T.
doi: 10.1007/s12104-021-10019-6
March 21, 2021

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

It is shown that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are very similar. In solution, however, the flexibility of the N-terminal helix-1 and loop from helix-1 to -2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between phosphorylated residues and the positively charged C-terminal helix-6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a huge loss of affinity for dsDNA, but does not impair binding to lamin A/C Igfold domain.

Marcelot A, Petitalot A, Ropars V, Le Du M-H, Samson C, Dubois S, Hoffmann G, Miron S, Cuniasse P, Marquez JA, Thai R, Theillet F-X, Zinn-Justin S.

doi: 10.1093/nar/gkab184

March 9, 2021

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

We recently reported an NMR bioreactor to maintain a high number of human cells metabolically active for up to 72 h. This setup was applied to monitor protein interactions and chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data. The method provides concentration profiles of the chemical species in the cells as a function of time, which can be used to obtain kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application.

Barbieri L, Luchinat E.

doi: 10.3791/62323


Cryo-focused ion beam lamella preparation protocol for in situ Structural Biology

The advances in cryo-EM enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since cryo-ET is generally limited to specimens with thickness <500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells was developed and optimized last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from vitrified cells. Preparation of lamellae from large adherent mammalian cells, a small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life-sciences.
Moravcová J, Dopitová R, Pinkas M, Nováček J.
doi: 10.1007/978-1-0716-1406-8

Magnetization transfer to enhance NOE cross‐peaks among labile protons: Applications to imino‐imino sequential walks in SARS‐CoV‐2‐derived RNAs

The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
Novakovic M, Kupce E, Scherf T, Oxenfarth A, Schnieders R, Grün T, Wirmer-Bartoschek J, Richter C, Schwalbe H, Frydman L.
doi: 10.1002/anie.202015948

Mixed-valence compounds as polarizing agents for Overhauser dynamic nuclear polarization in solids

In this paper a novel set of polarizing agents is tested by electron paramagnetic resonance methods and their performance si demonstrated in high-field Dynamic Nuclear Polarization (DNP) experiments in the solid state. The mixed-valence compounds used constitute a group of molecules, in which molecular mobility persists even in solids. Consequently, such polarizing agents can be used for Overhauser-DNP experiments in the solid-state, with favorable conditions for DNP at ultra-high magnetic fields.
Gurinov A, Sieland B, Kuzhelev A, Elgabarty H, Kuhne TD, Prisner T, Paradies J, Baldus M, Ivanov KL, Pylaeva S.

Protein in-cell NMR spectroscopy at 1.2 GHz

Here we show the first in-cell NMR spectra recorded at 1.2 GHz on human cells! We compare resolution and sensitivity against those obtained at 900 and 950 MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2 GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.
Luchinat E, Barbieri, L, Cremonini M, Banci L.
doi: 10.1007/s10858-021-00358-w

Real-time NMR spectroscopy in the study of biomolecular kinetics and dynamics

The review describes the application of NMR spectroscopy to study kinetics of folding, refolding and aggregation of proteins, RNA and DNA. In particular irreversible folding experiments pose large requirements on (i) the signal-to-noise due to the time limitations and (ii) on synchronizing the refolding steps. Here, we discuss the application of methods for signal-to-noise increases including DNP, hyperpolarization and photo-CIDNP for time-resolved NMR. Methods are reviewed ranging from pressure- and temperature-jump, light induction and rapid mixing to induce rapidly non-equilibrium conditions required to initiate folding.
Pintér G, Hohmann KF, Grün JT, Wirmer-Bartoschek J, Glaubitz C, Fürtig B, Schwalbe H.
doi: 10.5194/mr-2021-16

The folding landscapes of human telomeric RNA and DNA G-quadruplexes are markedly different

We investigated folding kinetics of G-quadruplex (G4) structures. In particular, we compare K+-induced folding of an RNA G4 derived from human telomeric repeat-containing RNA (TERRA25) with a homologous DNA G4 (wtTel25) using CD and real-time NMR spectroscopy. The folding kinetics of these G4's are markedly different: while DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favored off-pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs DNA G4s. Modified DNA G4s with 19F bound to C2’ in arabino configuration adopt exclusively anti conformations for chi. These fluoro-modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting correlation between folding kinetics and pathways with differences in chi-angle preferences in DNA and RNA, respectively.
Müller D, Bessi I, Richter C, Schwalbe H.
doi: 10.1002/anie.202100280

Facilitated crystal handling using a simple device for evaporation reduction in microtiter plates

Barthel T, Huschmann FU, Wallacher D, Feiler CG, Klebe G, Weiss MS, Wollenhaupt J.
doi: 10.1107/S1600576720016477
Oct 23, 2020

Structural rearrangement of amyloid-beta upon inhibitor binding suppresses formation of Alzheimer's disease related oligomers

Here, it is shown for two fibril inhibiting ligands - an ionic molecular tweezer and a hydrophobic peptide - that despite their different interaction mechanisms, the suppression of the fibril pathway can be deduced from the disappearance of the corresponding structure of the first amyloid-b oligomers.
Lieblein T, Zangl R, Martin J, Hoffmann J, Hutchison MJ, Stark T, Stirnal E, Schrader T, Schwalbe H, Morgner N.
doi: 10.7554/eLife.59306
Dec 14, 2020

Solution structure of the voltage-gated Tim23 channel in complex with a mitochondrial presequence peptide

Zhou S, Ruan M, Li Y, Yang J, Bai S, Richter C, Schwalbe H, Xie C, Shen B, Wang J.
doi: 10.1038/s41422-020-00452-y
Dec 10, 2020

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins

The Protein Ensemble Database (PED) holds structural ensembles of intrinsically disordered proteins. The new version, PED 4.0, has been redesigned and reimplemented with cutting-edge technology. It holds about six times more data and a broader representation of state-of-the-art ensemble generation methods than before, has a completely renewed graphical interface, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. A new submission process combines both automatic and manual evaluation steps, and a team of biocurators integrate structured metadata. A new search engine allows the user to build advanced queries and search all entry fields.
Lazar T, Martínez-Pérez E, Quaglia F, Hatos A, Chemes LB, Iserte JA, Méndez NA, Garrone NA, Saldaño TE, Marchetti J, Rueda AJV, Bernadó P, Blackledge M, Cordeiro TN, Fagerberg E, Forman-Kay JD, Fornasari MS, Gibson TJ, Gomes GW, Gradinaru CC, Head-Gordon T, Jensen MR, Lemke EA, Longhi S, Marino-Buslje C, Minervini G, Mittag T, Monzon AM, Pappu RV, Parisi G, Ricard-Blum S, Ruff KM, Salladini E, Skepö M, Svergun D, Vallet SD, Varadi M, Tompa P, Tosatto SCE, Piovesan D
doi: 10.1093/nar/gkaa1021
Oct 27, 2020

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). SEC-SAXS has become a well-established approach to reduce effects of sample heterogeneity on SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins.
Graewert MA, De Vela S, Gräwert TW, Molodenskiy DS, Blanchet CE, Svergun DI, Jeffries CM
doi: 10.3390/cryst10110975
Oct 16, 2020

Intracellular Binding/Unbinding Kinetics of Approved Drugs to Carbonic Anhydrase II Observed by in-Cell NMR

In-cell NMR was applied to investigate binding of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. We observed strikingly dose- and time-dependent binding, and some drugs exhibited a more complex behavior than others. Some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, thus preventing quantitative formation of a stable protein-ligand complex. This could be correlated to the off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design.
Luchinat E, Barbieri L, Cremonini M, Nocentini, A, Supuran CT, Banci L
doi: 10.1021/acschembio.0c00590
July 21, 2020

Real-time Quantitative In-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human Cells Using Multivariate Curve Resolution

Here, an improved bioreactor design is reported, which keeps human cells alive and metabolic for up to 72 h, as well as a novel workflow for quantitative analysis of real-time in-cell NMR data. We monitor protein-ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing fitting with kinetic models. The approach can be applied to study complex kinetic behavior of macromolecules in a cellular setting, and could be extended to any real-time NMR application in live cells.
Luchinat E, Barbieri L, Campbell TF, Banci L
doi: 10.1021/acs.analchem.0c01677
Sept 25, 2020

In-cell NMR of functional riboswitch aptamers in eukaryotic cells

We show the in-cell NMR-spectroscopic observation of the cognate ligand 2’-deoxyguanosine binding to the aptamer domain of the bacterial 2’-deoxyguanosine-sensing riboswitch in eukaryotic Xenopus laevis oocytes and in human HeLa cells. The binding mode established by in vitro characterization of this prokaryotic riboswitch appears maintained in eukaryotic cellular environment. Thus far, in-cell NMR studies on RNA in mammalian cells were limited to short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation. Now we show that in-cell NMR can be used for characterization of much larger (~70 nt) functional and chemically non-modified RNA.
Broft P, Dzatko S, Krafcikova M, Wacker A, Hansel-Hertsch R, Trantirek L, Schwalbe, H
doi: 10.1002/anie.202007184
Aug 14, 2020

19-F NMR-based fragment screening for 14 different biologically active RNAs and 10 DNA and protein counter-screens

Here the NMR 19F screening of RNA targets with different secondary and tertiary structure is shown to systematically assess the druggability of RNAs. Our RNAs include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. We show that RNA can be specifically targeted and to demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we show how to increase binding affinity from an initial hit using chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low micromolar binding affinity without losing binding specificity between two different terminator structures.
Binas O, de Jesus V, Landgraf T, Völklein AE, Martins J, Hymon D, Berg H, Bains JK, Biedenbänder T, Fürtig B, Gande SL, Niesteruk A, Oxenfarth A, Qureshi NS, Schamber T, Schnieders R, Tröster A, Wacker A, Wirmer-Bartoschek J, Martin MAW, Stirnal E, Azzaoui K, Blommers MJJ, Richter C, Sreeramulu S, Schwalbe H
doi: 10.1002/cbic.202000476

Anomeric Selectivity of Trehalose Transferase with Rare L-Sugars

Mestrom L, Marsden SR, Van der Eijk H, Laustsen JU, Jeffries CM, Svergun DI, Hagedoorn PL, Bento, I, Hanefeld, U
doi: 10.1021/acscatal.0c02117
July 17, 2020

Multitarget Virtual Screening for Drug Repurposing in COVID19

Sorzano CO, Crisman E, Carazo JM, Leon R
July 1, 2020

The basics of small-angle neutron scattering (SANS for new users of structural biology)

Jeffries CM, Pietras Z, Svergun DI
doi: 10.1051/epjconf/202023603001
May 24, 2020

Improvements on marker-free images alignment for electron tomography

Sorzano COS, De Isidro-Gomez F, Fernandez-Gimenez E, Herreros D, Marco S, Carazo JM, Messaoudi C
BioRxiv, doi: 10.1101/2020.05.22.110445
June 23, 2020

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Burda PC, Crosskey T, Lauk K, Zurborg A, Söhnchen C, Liffner B, Wilcke L, Pietsch E, Strauss J, Jeffries CM, Svergun DI, Wilson DW, Wilmanns M, Gilberger TW
doi: 10.1016/j.celrep.2020.107817
June 19, 2020

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy

Damman R, Lucini Paioni A, Xenaki KT, Beltrán Hernández I, van Bergen En Henegouwen PMP, Baldus M
doi: 10.1007/s10858-020-00328-8
June 12, 2020

NMR quality control of fragment libraries for screening

Sreeramulu S, Richter C, Kuehn T, Azzaoui K, Blommers MJJ, Del Conte R, Fragai M, Trieloff N, Schmieder P, Nazaré M, Specker E, Ivanov V, Oschkinat H, Banci L, Schwalbe H
doi: 10.1007/s10858-020-00327-9