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3860 KiB

Open AccessFeature PaperArticle

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion

by Natsuki Fukuda, Kentaro Noi, Lidong Weng, Yoshihiro Kobashigawa, Hiromi Miyazaki, Yukari Wakeyama, Michiyo Takaki, Yusuke Nakahara, Yuka Tatsuno, Makiyo Uchida-Kamekura, Yoshiaki Suwa, Takashi Sato, Naoki Ichikawa-Tomikawa, Motoyoshi Nomizu, Yukio Fujiwara, Fumina Ohsaka, Takashi Saitoh, Katsumi Maenaka, Hiroyuki Kumeta, Shoko Shinya, Chojiro Kojima, Teru Ogura and Hiroshi Morioka Show full author list Hide full author list

Molecules 2017, 22(10), 1695; https://doi.org/10.3390/molecules22101695 - 10 Oct 2017

Cited by 9 | Viewed by 6403

Abstract

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited [...] Read more.

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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2284 KiB

Open AccessArticle

NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

by Saeko Yanaka, Toshio Yamazaki, Rina Yogo, Masanori Noda, Susumu Uchiyama, Hirokazu Yagi and Koichi Kato

Molecules 2017, 22(10), 1619; https://doi.org/10.3390/molecules22101619 - 27 Sep 2017

Cited by 12 | Viewed by 5983

Abstract

Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures [...] Read more.

Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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6055 KiB

Open AccessArticle

The Recognition of Calmodulin to the Target Sequence of Calcineurin—A Novel Binding Mode

by Chia-Lin Chyan, Deli Irene and Sin-Mao Lin

Molecules 2017, 22(10), 1584; https://doi.org/10.3390/molecules22101584 - 21 Sep 2017

Cited by 7 | Viewed by 5896

Abstract

Calcineurin (CaN) is a Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase, which plays essential roles in many cellular and developmental processes. CaN comprises two subunits, a catalytic subunit (CaN-A, 60 kDa) and a regulatory subunit (CaN-B, 19 kDa). CaN-A tightly binds to CaN-B in [...] Read more.

Calcineurin (CaN) is a Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase, which plays essential roles in many cellular and developmental processes. CaN comprises two subunits, a catalytic subunit (CaN-A, 60 kDa) and a regulatory subunit (CaN-B, 19 kDa). CaN-A tightly binds to CaN-B in the presence of minimal levels of Ca²⁺, but the enzyme is inactive until activated by CaM. Upon binding to CaM, CaN then undergoes a conformational rearrangement, the auto inhibitory domain is displaced and thus allows for full activity. In order to elucidate the regulatory role of CaM in the activation processes of CaN, we used NMR spectroscopy to determine the structure of the complex of CaM and the target peptide of CaN (CaNp). The CaM/CaNp complex shows a compact ellipsoidal shape with 8 α-helices of CaM wrapping around the CaNp helix. The RMSD of backbone and heavy atoms of twenty lowest energy structures of CaM/CaNp complex are 0.66 and 1.14 Å, respectively. The structure of CaM/CaNp complex can be classified as a novel binding mode family 1–18 with major anchor residues Ile³⁹⁶ and Leu⁴¹³ to allocate the largest space between two domains of CaM. The relative orientation of CaNp to CaM is similar to the CaMKK peptide in the 1–16 binding mode with N- and C-terminal hydrophobic anchors of target sequence engulfed in the hydrophobic pockets of the N- and C-domain of CaM, respectively. In the light of the structural model of CaM/CaNp complex reported here, we provide new insight in the activation processes of CaN by CaM. We propose that the hydrophobic interactions between the Ca²⁺-saturated C-domain and C-terminal half of the target sequence provide driving forces for the initial recognition. Subsequent folding in the target sequence and structural readjustments in CaM enhance the formation of the complex and affinity to calcium. The electrostatic repulsion between CaM/CaNp complex and AID may result in the displacement of AID from active site for full activity. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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1573 KiB

Open AccessCommunication

Forbidden Coherence Transfer of ¹⁹F Nuclei to Quantitatively Measure the Dynamics of a CF₃-Containing Ligand in Receptor-Bound States

by Yuji Tokunaga, Koh Takeuchi and Ichio Shimada

Molecules 2017, 22(9), 1492; https://doi.org/10.3390/molecules22091492 - 7 Sep 2017

Cited by 4 | Viewed by 5407

Abstract

The dynamic property of a ligand in the receptor-bound state is an important metric to characterize the interactions in the ligand–receptor interface, and the development of an experimental strategy to quantify the amplitude of motions in the bound state is of importance to [...] Read more.

The dynamic property of a ligand in the receptor-bound state is an important metric to characterize the interactions in the ligand–receptor interface, and the development of an experimental strategy to quantify the amplitude of motions in the bound state is of importance to introduce the dynamic aspect into structure-guided drug development (SGDD). Fluorine modifications are frequently introduced at the hit-to-lead optimization stage to enhance the binding potency and other characteristics of a ligand. However, the effects of fluorine modifications are generally difficult to predict, owing to the pleiotropic nature of the interactions. In this study, we report an NMR-based approach to experimentally evaluate the local dynamics of trifluoromethyl (CF₃)-containing ligands in the receptor-bound states. For this purpose, the forbidden coherence transfer (FCT) analysis, which has been used to study the dynamics of methyl moieties in proteins, was extended to the ¹⁹F nuclei of CF₃-containing ligands. By applying this CF₃–FCT analysis to a model interaction system consisting of a ligand, AST-487, and a receptor, p38α, we successfully quantified the amplitude of the CF₃ dynamics in the p38α-bound state. The strategy would bring the CF₃-containing ligands within the scope of dynamic SGDD to improve the affinity and specificity for the drug-target receptors. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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5778 KiB

Open AccessArticle

Interactions Controlling the Slow Dynamic Conformational Motions of Ubiquitin

by Soichiro Kitazawa, Maho Yagi-Utsumi, Koichi Kato and Ryo Kitahara

Molecules 2017, 22(9), 1414; https://doi.org/10.3390/molecules22091414 - 28 Aug 2017

Cited by 4 | Viewed by 4728

Abstract

Rational mutation of proteins based on their structural and dynamic characteristics is a useful strategy for amplifying specific fluctuations in proteins. Here, we show the effects of mutation on the conformational fluctuations and thermodynamic stability of ubiquitin. In particular, we focus on the [...] Read more.

Rational mutation of proteins based on their structural and dynamic characteristics is a useful strategy for amplifying specific fluctuations in proteins. Here, we show the effects of mutation on the conformational fluctuations and thermodynamic stability of ubiquitin. In particular, we focus on the salt bridge between K11 and E34 and the hydrogen bond between I36 and Q41, which are predicted to control the fluctuation between the basic folded state, N₁, and the alternatively folded state, N₂, of the protein, using high-pressure NMR spectroscopy. The E34A mutation, which disrupts the salt bridge, did not alter picosecond–to–nanosecond, microsecond–to–millisecond dynamic motions, and stability of the protein, while the Q41N mutation, which destabilizes the hydrogen bond, specifically amplified the N₁–N₂ conformational fluctuation and decreased stability. Based on the observed thermodynamic stabilities of the various conformational states, we showed that in the Q41N mutant, the N₁ state is more significantly destabilized than the N₂ state, resulting in an increase in the relative population of N₂. Identifying the interactions controlling specific motions of a protein will facilitate molecular design to achieve functional dynamics beyond native state dynamics. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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3688 KiB

Open AccessFeature PaperArticle

Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen–Deuterium Exchange

by Chih-Ta Henry Chien, Meng-Ru Ho, Chung-Hung Lin and Shang-Te Danny Hsu

Molecules 2017, 22(8), 1357; https://doi.org/10.3390/molecules22081357 - 16 Aug 2017

Cited by 15 | Viewed by 6577

Abstract

Galectins are β-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose [...] Read more.

Galectins are β-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8^NTD and hGal8^CTD, respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8^NTD. We further demonstrated using the NMR-based hydrogen–deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8^NTD, indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8^CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8^CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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1137 KiB

Open AccessArticle

A Unique and Simple Approach to Improve Sensitivity in ¹⁵N-NMR Relaxation Measurements for NH₃⁺ Groups: Application to a Protein-DNA Complex

by Dan Nguyen, Ganesh L. R. Lokesh, David E. Volk and Junji Iwahara

Molecules 2017, 22(8), 1355; https://doi.org/10.3390/molecules22081355 - 15 Aug 2017

Cited by 5 | Viewed by 5474

Abstract

NMR spectroscopy is a powerful tool for research on protein dynamics. In the past decade, there has been significant progress in the development of NMR methods for studying charged side chains. In particular, NMR methods for lysine side-chain NH₃⁺ groups have [...] Read more.

NMR spectroscopy is a powerful tool for research on protein dynamics. In the past decade, there has been significant progress in the development of NMR methods for studying charged side chains. In particular, NMR methods for lysine side-chain NH₃⁺ groups have been proven to be powerful for investigating the dynamics of hydrogen bonds or ion pairs that play important roles in biological processes. However, relatively low sensitivity has been a major practical issue in NMR experiments on NH₃⁺ groups. In this paper, we present a unique and simple approach to improve sensitivity in ¹⁵N relaxation measurements for NH₃⁺ groups. In this approach, the efficiency of coherence transfers for the desired components are maximized, whereas undesired anti-phase or multi-spin order components are purged through pulse schemes and rapid relaxation. For lysine side-chain NH₃⁺ groups of a protein-DNA complex, we compared the data obtained with the previous and new pulse sequences under the same conditions and confirmed that the ¹⁵N relaxation parameters were consistent for these datasets. While retaining accuracy in measuring ¹⁵N relaxation, our new pulse sequences for NH₃⁺ groups allowed an 82% increase in detection sensitivity of ¹⁵N longitudinal and transverse relaxation measurements. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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698 KiB

Open AccessArticle

Capillary-Inserted Rotor Design for HRµMAS NMR-Based Metabolomics on Mass-Limited Neurospheres

by Nghia Tuan Duong, Masanori Yamato, Masayuki Nakano, Satoshi Kume, Yasuhisa Tamura, Yosky Kataoka, Alan Wong and Yusuke Nishiyama

Molecules 2017, 22(8), 1289; https://doi.org/10.3390/molecules22081289 - 3 Aug 2017

Cited by 4 | Viewed by 5189

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique and has been widely used in metabolomics. However, the intrinsic low sensitivity of NMR prevents its applications to systems with limited sample availabilities. In this study, a new experimental approach is presented to [...] Read more.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique and has been widely used in metabolomics. However, the intrinsic low sensitivity of NMR prevents its applications to systems with limited sample availabilities. In this study, a new experimental approach is presented to analyze mass-scarce samples in limited volumes of less than 300 nL with simple handling. The sample is loaded into the glass capillary, and this capillary is then inserted into a Kel-F rotor. The experimental performance of the capillary-inserted rotor (capillary-insert) is investigated on an isotropic solution of sucrose by the use of a high-resolution micro-sized magic angle spinning (HRµMAS) probe. The acquired NMR signal’s sensitivity to a given sample amount is comparable or even higher in comparison to that recorded by the standard solution NMR probe. More importantly, this capillary-insert coupled with the HRµMAS probe allows in-depth studies of heterogeneous samples as the MAS removes the line broadening caused by the heterogeneity. The NMR analyses of mass-limited cultured neurospheres have been demonstrated, resulting in high quality spectra where numerous metabolites are unambiguously identified. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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4798 KiB

Open AccessArticle

Effects of K11R and G31P Mutations on the Structure and Biological Activities of CXCL8: Solution Structure of Human CXCL8_(3-72)K11R/G31P

by Hsi-Tsung Cheng, Hui-Yuan Yu, John R. Gordon, Fang Li and Jya-Wei Cheng

Molecules 2017, 22(7), 1229; https://doi.org/10.3390/molecules22071229 - 21 Jul 2017

Cited by 5 | Viewed by 5318

Abstract

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), the expression levels of which are elevated in many inflammatory diseases, binds to both the CXCR1 and CXCR2 receptors with high affinity. Recently, an [...] Read more.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), the expression levels of which are elevated in many inflammatory diseases, binds to both the CXCR1 and CXCR2 receptors with high affinity. Recently, an analogue of human CXCL8, CXCL8_(3–72)K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both the CXCR1 and CXCR2. Herein, we have determined the solution structure and the CXCR1 N-terminal peptide binding sites of hG31P by NMR spectroscopy. We have found that the displacement within the tertiary structure of the 30 s loop and the N-terminal region and more specifically change of the loop conformation (especially H33), of hG31P may affect its binding to the CXCR1 receptor and thereby inhibit human neutrophil chemotactic responses induced by ELR-CXC chemokines. Our results provide a structural basis for future clinical investigations of this CXCR1/CXCR2 receptor antagonist and for the further development of CXCL8 based antagonists. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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3822 KiB

Open AccessArticle

Structural Insight into the Recognition of r(UAG) by Musashi-1 RBD2, and Construction of a Model of Musashi-1 RBD1-2 Bound to the Minimum Target RNA

by Ryo Iwaoka, Takashi Nagata, Kengo Tsuda, Takao Imai, Hideyuki Okano, Naohiro Kobayashi and Masato Katahira

Molecules 2017, 22(7), 1207; https://doi.org/10.3390/molecules22071207 - 19 Jul 2017

Cited by 23 | Viewed by 7236

Abstract

Musashi-1 (Msi1) controls the maintenance of stem cells and tumorigenesis through binding to its target mRNAs and subsequent translational regulation. Msi1 has two RNA-binding domains (RBDs), RBD1 and RBD2, which recognize r(GUAG) and r(UAG), respectively. These minimal recognition sequences are connected by variable [...] Read more.

Musashi-1 (Msi1) controls the maintenance of stem cells and tumorigenesis through binding to its target mRNAs and subsequent translational regulation. Msi1 has two RNA-binding domains (RBDs), RBD1 and RBD2, which recognize r(GUAG) and r(UAG), respectively. These minimal recognition sequences are connected by variable linkers in the Msi1 target mRNAs, however, the molecular mechanism by which Msi1 recognizes its targets is not yet understood. We previously determined the solution structure of the Msi1 RBD1:r(GUAGU) complex. Here, we determined the first structure of the RBD2:r(GUAGU) complex. The structure revealed that the central trinucleotide, r(UAG), is specifically recognized by the intermolecular hydrogen-bonding and aromatic stacking interactions. Importantly, the C-terminal region, which is disordered in the free form, took a certain conformation, resembling a helix. The observation of chemical shift perturbation and intermolecular NOEs, together with increases in the heteronuclear steady-state {¹H}-¹⁵N NOE values on complex formation, indicated the involvement of the C-terminal region in RNA binding. On the basis of the two complex structures, we built a structural model of consecutive RBDs with r(UAGGUAG) containing both minimal recognition sequences, which resulted in no steric hindrance. The model suggests recognition of variable lengths (n) of the linker up to n = 50 may be possible. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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3539 KiB

Open AccessArticle

Dynamic Allostery Modulates Catalytic Activity by Modifying the Hydrogen Bonding Network in the Catalytic Site of Human Pin1

by Jing Wang, Ryosuke Kawasaki, Jun-ichi Uewaki, Arif U. R. Rashid, Naoya Tochio and Shin-ichi Tate

Molecules 2017, 22(6), 992; https://doi.org/10.3390/molecules22060992 - 15 Jun 2017

Cited by 13 | Viewed by 5418

Abstract

Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a [...] Read more.

Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a WW domain for substrate recognition and a peptidyl-prolyl isomerase (PPIase) domain. Previous studies of Pin1 showed that physical contact between the domains causes dynamic allostery by reducing conformation dynamics in the catalytic domain, which compensates for the entropy costs of substrate binding to the catalytic site and thus increases catalytic activity. In this study, the S138A mutant PPIase domain, a mutation that mimics the structural impact of the interdomain contact, was demonstrated to display dynamic allostery by rigidification of the α2-α3 loop that harbors the key catalytic residue C113. The reduced dynamics of the α2-α3 loop stabilizes the C113–H59 hydrogen bond in the hydrogen-bonding network of the catalytic site. The stabilized hydrogen bond between C113 and H59 retards initiation of isomerization, which explains the reduced isomerization rate by ~20% caused by the S138A mutation. These results provide new insight into the interdomain allosteric communication of Pin1. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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Review

Jump to: Research

27 pages, 5329 KiB

Open AccessReview

NMR-Fragment Based Virtual Screening: A Brief Overview

by Meenakshi Singh, Benjamin Tam and Barak Akabayov

Molecules 2018, 23(2), 233; https://doi.org/10.3390/molecules23020233 - 25 Jan 2018

Cited by 38 | Viewed by 9968

Abstract

Fragment-based drug discovery (FBDD) using NMR has become a central approach over the last twenty years for development of small molecule inhibitors against biological macromolecules, to control a variety of cellular processes. Yet, several considerations should be taken into account for obtaining a [...] Read more.

Fragment-based drug discovery (FBDD) using NMR has become a central approach over the last twenty years for development of small molecule inhibitors against biological macromolecules, to control a variety of cellular processes. Yet, several considerations should be taken into account for obtaining a therapeutically relevant agent. In this review, we aim to list the considerations that make NMR fragment screening a successful process for yielding potent inhibitors. Factors that may govern the competence of NMR in fragment based drug discovery are discussed, as well as later steps that involve optimization of hits obtained by NMR-FBDD. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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27 pages, 2394 KiB

Open AccessReview

Current NMR Techniques for Structure-Based Drug Discovery

by Toshihiko Sugiki, Kyoko Furuita, Toshimichi Fujiwara and Chojiro Kojima

Molecules 2018, 23(1), 148; https://doi.org/10.3390/molecules23010148 - 12 Jan 2018

Cited by 96 | Viewed by 14530

Abstract

A variety of nuclear magnetic resonance (NMR) applications have been developed for structure-based drug discovery (SBDD). NMR provides many advantages over other methods, such as the ability to directly observe chemical compounds and target biomolecules, and to be used for ligand-based and protein-based [...] Read more.

A variety of nuclear magnetic resonance (NMR) applications have been developed for structure-based drug discovery (SBDD). NMR provides many advantages over other methods, such as the ability to directly observe chemical compounds and target biomolecules, and to be used for ligand-based and protein-based approaches. NMR can also provide important information about the interactions in a protein-ligand complex, such as structure, dynamics, and affinity, even when the interaction is too weak to be detected by ELISA or fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) or to be crystalized. In this study, we reviewed current NMR techniques. We focused on recent progress in NMR measurement and sample preparation techniques that have expanded the potential of NMR-based SBDD, such as fluorine NMR (¹⁹F-NMR) screening, structure modeling of weak complexes, and site-specific isotope labeling of challenging targets. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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3806 KiB

Open AccessFeature PaperReview

Solution NMR Studies of Mycobacterium tuberculosis Proteins for Antibiotic Target Discovery

by Do-Hee Kim, Sung-Min Kang and Bong-Jin Lee

Molecules 2017, 22(9), 1447; https://doi.org/10.3390/molecules22091447 - 31 Aug 2017

Cited by 2 | Viewed by 5323

Abstract

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which triggers severe pulmonary diseases. Recently, multidrug/extensively drug-resistant tuberculosis strains have emerged and continue to threaten global health. Because of the development of drug-resistant tuberculosis, there is an urgent need for novel antibiotics [...] Read more.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which triggers severe pulmonary diseases. Recently, multidrug/extensively drug-resistant tuberculosis strains have emerged and continue to threaten global health. Because of the development of drug-resistant tuberculosis, there is an urgent need for novel antibiotics to treat these drug-resistant bacteria. In light of the clinical importance of M. tuberculosis, 2067 structures of M. tuberculsosis proteins have been determined. Among them, 52 structures have been solved and studied using solution nuclear magnetic resonance (NMR). The functional details based on structural analysis of M. tuberculosis using NMR can provide essential biochemical data for the development of novel antibiotic drugs. In this review, we introduce diverse structural and biochemical studies on M. tuberculosis proteins determined using NMR spectroscopy. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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2692 KiB

Open AccessReview

Solution NMR Spectroscopy in Target-Based Drug Discovery

by Yan Li and Congbao Kang

Molecules 2017, 22(9), 1399; https://doi.org/10.3390/molecules22091399 - 23 Aug 2017

Cited by 31 | Viewed by 9947

Abstract

Solution NMR spectroscopy is a powerful tool to study protein structures and dynamics under physiological conditions. This technique is particularly useful in target-based drug discovery projects as it provides protein-ligand binding information in solution. Accumulated studies have shown that NMR will play more [...] Read more.

Solution NMR spectroscopy is a powerful tool to study protein structures and dynamics under physiological conditions. This technique is particularly useful in target-based drug discovery projects as it provides protein-ligand binding information in solution. Accumulated studies have shown that NMR will play more and more important roles in multiple steps of the drug discovery process. In a fragment-based drug discovery process, ligand-observed and protein-observed NMR spectroscopy can be applied to screen fragments with low binding affinities. The screened fragments can be further optimized into drug-like molecules. In combination with other biophysical techniques, NMR will guide structure-based drug discovery. In this review, we describe the possible roles of NMR spectroscopy in drug discovery. We also illustrate the challenges encountered in the drug discovery process. We include several examples demonstrating the roles of NMR in target-based drug discoveries such as hit identification, ranking ligand binding affinities, and mapping the ligand binding site. We also speculate the possible roles of NMR in target engagement based on recent processes in in-cell NMR spectroscopy. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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4547 KiB

Open AccessReview

Application of Solution NMR to Structural Studies on α-Helical Integral Membrane Proteins

by Dae-Won Sim, Zhenwei Lu, Hyung-Sik Won, Seu-Na Lee, Min-Duk Seo, Bong-Jin Lee and Ji-Hun Kim

Molecules 2017, 22(8), 1347; https://doi.org/10.3390/molecules22081347 - 15 Aug 2017

Cited by 12 | Viewed by 7681

Abstract

A large portion of proteins in living organisms are membrane proteins which play critical roles in the biology of the cell, from maintenance of the biological membrane integrity to communication of cells with their surroundings. To understand their mechanism of action, structural information [...] Read more.

A large portion of proteins in living organisms are membrane proteins which play critical roles in the biology of the cell, from maintenance of the biological membrane integrity to communication of cells with their surroundings. To understand their mechanism of action, structural information is essential. Nevertheless, structure determination of transmembrane proteins is still a challenging area, even though recently the number of deposited structures of membrane proteins in the PDB has rapidly increased thanks to the efforts using X-ray crystallography, electron microscopy, and solid and solution nuclear magnetic resonance (NMR) technology. Among these technologies, solution NMR is a powerful tool for studying protein-protein, protein-ligand interactions and protein dynamics at a wide range of time scales as well as structure determination of membrane proteins. This review provides general and useful guideline for membrane protein sample preparation and the choice of membrane-mimetic media, which are the key step for successful structural analysis. Furthermore, this review provides an opportunity to look at recent applications of solution NMR to structural studies on α-helical membrane proteins through some success stories. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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7235 KiB

Open AccessFeature PaperReview

The Exact Nuclear Overhauser Enhancement: Recent Advances

by Parker J. Nichols, Alexandra Born, Morkos A. Henen, Dean Strotz, Julien Orts, Simon Olsson, Peter Güntert, Celestine N. Chi and Beat Vögeli

Molecules 2017, 22(7), 1176; https://doi.org/10.3390/molecules22071176 - 14 Jul 2017

Cited by 21 | Viewed by 6935

Abstract

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures [...] Read more.

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions. Full article

(This article belongs to the Special Issue Recent Advances in Biomolecular NMR Spectroscopy)

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