Research

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12 pages, 1577 KiB

Open AccessArticle

In Situ Monitoring of Bacteria under Antimicrobial Stress Using ³¹P Solid-State NMR

by Sarah A. Overall, Shiying Zhu, Eric Hanssen, Frances Separovic and Marc-Antoine Sani

Int. J. Mol. Sci. 2019, 20(1), 181; https://doi.org/10.3390/ijms20010181 - 06 Jan 2019

Cited by 33 | Viewed by 4286

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. [...] Read more.

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that ³¹P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although ³¹P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of ³¹P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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14 pages, 2923 KiB

Open AccessArticle

In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau

by Shengnan Zhang, Chuchu Wang, Jinxia Lu, Xiaojuan Ma, Zhenying Liu, Dan Li, Zhijun Liu and Cong Liu

Int. J. Mol. Sci. 2019, 20(1), 90; https://doi.org/10.3390/ijms20010090 - 26 Dec 2018

Cited by 19 | Viewed by 5728

Abstract

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the [...] Read more.

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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17 pages, 2943 KiB

Open AccessArticle

Unambiguous Ex Situ and in Cell 2D ¹³C Solid-State NMR Characterization of Starch and Its Constituents

by Alexandre Poulhazan, Alexandre A. Arnold, Dror E. Warschawski and Isabelle Marcotte

Int. J. Mol. Sci. 2018, 19(12), 3817; https://doi.org/10.3390/ijms19123817 - 30 Nov 2018

Cited by 24 | Viewed by 4497

Abstract

Starch is the most abundant energy storage molecule in plants and is an essential part of the human diet. This glucose polymer is composed of amorphous and crystalline domains in different forms (A and B types) with specific physicochemical properties that determine its [...] Read more.

Starch is the most abundant energy storage molecule in plants and is an essential part of the human diet. This glucose polymer is composed of amorphous and crystalline domains in different forms (A and B types) with specific physicochemical properties that determine its bioavailability for an organism, as well as its value in the food industry. Using two-dimensional (2D) high resolution solid-state nuclear magnetic resonance (SS-NMR) on ¹³C-labelled starches that were obtained from Chlamydomonas reinhardtii microalgae, we established a complete and unambiguous assignment for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. We also assigned so far unreported non-reducing end groups and assessed starch chain length, crystallinity and amylose content. Starch was then characterized in situ, i.e., by ¹³C solid-state NMR of intact microalgal cells. Our in-cell methodology also enabled the identification of the effect of nitrogen starvation on starch metabolism. This work shows how solid-state NMR can enable the identification of starch structure, chemical modifications and biosynthesis in situ in intact microorganisms, eliminating time consuming and potentially altering purification steps. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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Review

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13 pages, 3536 KiB

Open AccessReview

Protein Structure Determination in Living Cells

by Teppei Ikeya, Peter Güntert and Yutaka Ito

Int. J. Mol. Sci. 2019, 20(10), 2442; https://doi.org/10.3390/ijms20102442 - 17 May 2019

Cited by 21 | Viewed by 4088

Abstract

To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes [...] Read more.

To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes but not ‘direct’ data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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21 pages, 10441 KiB

Open AccessReview

The Inescapable Effects of Ribosomes on In-Cell NMR Spectroscopy and the Implications for Regulation of Biological Activity

by David S. Burz, Leonard Breindel and Alexander Shekhtman

Int. J. Mol. Sci. 2019, 20(6), 1297; https://doi.org/10.3390/ijms20061297 - 14 Mar 2019

Cited by 4 | Viewed by 3256

Abstract

The effects of RNA on in-cell NMR spectroscopy and ribosomes on the kinetic activity of several metabolic enzymes are reviewed. Quinary interactions between labelled target proteins and RNA broaden in-cell NMR spectra yielding apparent megadalton molecular weights in-cell. The in-cell spectra can be [...] Read more.

The effects of RNA on in-cell NMR spectroscopy and ribosomes on the kinetic activity of several metabolic enzymes are reviewed. Quinary interactions between labelled target proteins and RNA broaden in-cell NMR spectra yielding apparent megadalton molecular weights in-cell. The in-cell spectra can be resolved by using cross relaxation-induced polarization transfer (CRINEPT), heteronuclear multiple quantum coherence (HMQC), transverse relaxation-optimized, NMR spectroscopy (TROSY). The effect is reproduced in vitro by using reconstituted total cellular RNA and purified ribosome preparations. Furthermore, ribosomal binding antibiotics alter protein quinary structure through protein-ribosome and protein-mRNA-ribosome interactions. The quinary interactions of Adenylate kinase, Thymidylate synthase and Dihydrofolate reductase alter kinetic properties of the enzymes. The results demonstrate that ribosomes may specifically contribute to the regulation of biological activity. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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19 pages, 265 KiB

Open AccessReview

Quo Vadis Biomolecular NMR Spectroscopy?

by Philipp Selenko

Int. J. Mol. Sci. 2019, 20(6), 1278; https://doi.org/10.3390/ijms20061278 - 14 Mar 2019

Cited by 14 | Viewed by 4635

Abstract

In-cell nuclear magnetic resonance (NMR) spectroscopy offers the possibility to study proteins and other biomolecules at atomic resolution directly in cells. As such, it provides compelling means to complement existing tools in cellular structural biology. Given the dominance of electron microscopy (EM)-based methods [...] Read more.

In-cell nuclear magnetic resonance (NMR) spectroscopy offers the possibility to study proteins and other biomolecules at atomic resolution directly in cells. As such, it provides compelling means to complement existing tools in cellular structural biology. Given the dominance of electron microscopy (EM)-based methods in current structure determination routines, I share my personal view about the role of biomolecular NMR spectroscopy in the aftermath of the revolution in resolution. Specifically, I focus on spin-off applications that in-cell NMR has helped to develop and how they may provide broader and more generally applicable routes for future NMR investigations. I discuss the use of ‘static’ and time-resolved solution NMR spectroscopy to detect post-translational protein modifications (PTMs) and to investigate structural consequences that occur in their response. I argue that available examples vindicate the need for collective and systematic efforts to determine post-translationally modified protein structures in the future. Furthermore, I explain my reasoning behind a Quinary Structure Assessment (QSA) initiative to interrogate cellular effects on protein dynamics and transient interactions present in physiological environments. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

23 pages, 2109 KiB

Open AccessReview

In-Cell NMR: Analysis of Protein–Small Molecule Interactions, Metabolic Processes, and Protein Phosphorylation

by Amit Kumar, Lars T. Kuhn and Jochen Balbach

Int. J. Mol. Sci. 2019, 20(2), 378; https://doi.org/10.3390/ijms20020378 - 17 Jan 2019

Cited by 12 | Viewed by 6704

Abstract

Nuclear magnetic resonance (NMR) spectroscopy enables the non-invasive observation of biochemical processes, in living cells, at comparably high spectral and temporal resolution. Preferably, means of increasing the detection limit of this powerful analytical method need to be applied when observing cellular processes under [...] Read more.

Nuclear magnetic resonance (NMR) spectroscopy enables the non-invasive observation of biochemical processes, in living cells, at comparably high spectral and temporal resolution. Preferably, means of increasing the detection limit of this powerful analytical method need to be applied when observing cellular processes under physiological conditions, due to the low sensitivity inherent to the technique. In this review, a brief introduction to in-cell NMR, protein–small molecule interactions, posttranslational phosphorylation, and hyperpolarization NMR methods, used for the study of metabolites in cellulo, are presented. Recent examples of method development in all three fields are conceptually highlighted, and an outlook into future perspectives of this emerging area of NMR research is given. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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19 pages, 1518 KiB

Open AccessReview

Applications of In-Cell NMR in Structural Biology and Drug Discovery

by CongBao Kang

Int. J. Mol. Sci. 2019, 20(1), 139; https://doi.org/10.3390/ijms20010139 - 02 Jan 2019

Cited by 35 | Viewed by 6454

Abstract

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions [...] Read more.

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement. Full article

(This article belongs to the Special Issue In-Cell NMR Spectroscopy: Biomolecular Structure and Function)

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