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NMR Tools and Their Application for Structural Biology of Bioactive Proteins

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 18881

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Dr. Orsolya Tőke

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Guest Editor

Research Centre for Natural Sciences, Centre for Structural Science, H-1117 Budapest, Hungary
Interests: protein structure and dynamics; protein-lipid interactions; cellular signaling; membrane peptides; protein folding; NMR spectroscopy
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Gyula Batta

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Faculty of Science and Technology, Institute of Chemistry, Department of Organic Chemistry, University of Debrecen, H-4032 Debrecen, Hungary
Interests: antibacterial activity; protein structures; antimicrobial proteins; glycopeptides
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

NMR spectroscopy provides structural and dynamic information at the atomic level, providing insight into molecular recognition mechanisms in proteins. Methodological and instrumentational developments in conjunction with inventive labeling strategies are pushing the limit of molecular weight and the complexity of systems suitable for investigation in both the solution and solid states. One of the main advantages of NMR is that in addition to providing us with high-resolution structural information, it has the capability to sample molecular motions on a wide range of timescales. Unveiling functionally relevant low-populated higher energy conformations, the characterization of oligomeric and aggregated protein states, and uncovering the relationship between folding and binding in intrinsically disordered proteins are only a few of the widely recognized contributions of NMR with pharmaceutical implications.

This Special Issue invites original research papers and reviews in the field of solution- and solid-state NMR devoted to deciphering the mechanism of action of proteins in bacterial and eukaryotic systems to improve our understanding of pathologic conditions with the hope of contributing to the fight against currently incurable disease states.

Dr. Orsolya Tőke
Prof. Dr. Gyula Batta
Guest Editors

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Keywords

protein interactions
ligand binding
molecular recognition
conformational transitions
allostery
intrinsically disordered proteins
protein dynamics
protein aggregation
membrane proteins
biofilms

Published Papers (11 papers)

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Editorial

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6 pages, 204 KiB

Open AccessEditorial

Dynamic Structures of Bioactive Proteins as Determined by Nuclear Magnetic Resonance

by Orsolya Toke and Gyula Batta

Int. J. Mol. Sci. 2024, 25(1), 295; https://doi.org/10.3390/ijms25010295 - 25 Dec 2023

Viewed by 667

Abstract

According to “Panta rhei”, a phrase by the ancient Greeks, you cannot enter the same river two times [...] Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

Research

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17 pages, 3432 KiB

Open AccessArticle

NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg²⁺ Ion

by Márton Gadanecz, Zsolt Fazekas, Gyula Pálfy, Dóra Karancsiné Menyhárd and András Perczel

Int. J. Mol. Sci. 2023, 24(15), 12101; https://doi.org/10.3390/ijms241512101 - 28 Jul 2023

Cited by 1 | Viewed by 985

Abstract

In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg²⁺-free and Mg²⁺-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg²⁺-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg²⁺-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an “NMR ensemble” based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg²⁺-bound and Mg²⁺-free states of G12C KRAS/GDP. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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13 pages, 2225 KiB

Open AccessArticle

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study

by András Czajlik, Ágnes Batta, Kinga Kerner, Ádám Fizil, Dorottya Hajdu, Mária Raics, Katalin E. Kövér and Gyula Batta

Int. J. Mol. Sci. 2023, 24(2), 1208; https://doi.org/10.3390/ijms24021208 - 7 Jan 2023

Cited by 2 | Viewed by 1604

Abstract

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3–4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAF^D19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated ¹⁵N-¹H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H₂O mixtures were more similar in the mixed solvents then they were in water. The ¹⁵N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the T_m melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAF^D19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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12 pages, 2968 KiB

Open AccessArticle

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

by Vladislav A. Lushpa, Nadezhda S. Baleeva, Sergey A. Goncharuk, Marina V. Goncharuk, Alexander S. Arseniev, Mikhail S. Baranov and Konstantin S. Mineev

Int. J. Mol. Sci. 2022, 23(19), 11361; https://doi.org/10.3390/ijms231911361 - 26 Sep 2022

Cited by 2 | Viewed by 1279

Abstract

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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24 pages, 10896 KiB

Open AccessArticle

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein

by Gergő Horváth, Bence Balterer, András Micsonai, József Kardos and Orsolya Toke

Int. J. Mol. Sci. 2022, 23(19), 11346; https://doi.org/10.3390/ijms231911346 - 26 Sep 2022

Cited by 1 | Viewed by 1231

Abstract

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling βD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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15 pages, 3901 KiB

Open AccessArticle

Inhibition of FGFR Signaling by Targeting FGF/FGFR Extracellular Interactions: Towards the Comprehension of the Molecular Mechanism through NMR Approaches

by Katiuscia Pagano, Elisa Longhi, Henriette Molinari, Giulia Taraboletti and Laura Ragona

Int. J. Mol. Sci. 2022, 23(18), 10860; https://doi.org/10.3390/ijms231810860 - 17 Sep 2022

Cited by 2 | Viewed by 1810

Abstract

NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein–protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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14 pages, 2306 KiB

Open AccessArticle

The Structural Flexibility of PR-10 Food Allergens

by Sebastian Führer, Jana Unterhauser, Ricarda Zeindl, Reiner Eidelpes, Monica L. Fernández-Quintero, Klaus R. Liedl and Martin Tollinger

Int. J. Mol. Sci. 2022, 23(15), 8252; https://doi.org/10.3390/ijms23158252 - 26 Jul 2022

Cited by 12 | Viewed by 2548

Abstract

PR-10 proteins constitute a major cause of food allergic reactions. Birch-pollen-related food allergies are triggered by the immunologic cross-reactivity of IgE antibodies with structurally homologous PR-10 proteins that are present in birch pollen and various food sources. While the three-dimensional structures of PR-10 food allergens have been characterized in detail, only a few experimental studies have addressed the structural flexibility of these proteins. In this study, we analyze the millisecond-timescale structural flexibility of thirteen PR-10 proteins from prevalent plant food sources by NMR relaxation-dispersion spectroscopy, in a comparative manner. We show that all the allergens in this study have inherently flexible protein backbones in solution, yet the extent of the structural flexibility appears to be strikingly protein-specific (but not food-source-specific). Above-average flexibility is present in the two short helices, α1 and α2, which form a V-shaped support for the long C-terminal helix α3, and shape the internal ligand-binding cavity, which is characteristic for PR-10 proteins. An in-depth analysis of the NMR relaxation-dispersion data for the PR-10 allergen from peanut reveals the presence of at least two subglobal conformational transitions on the millisecond timescale, which may be related to the release of bound low-molecular-weight ligands from the internal cavity. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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21 pages, 5792 KiB

Open AccessArticle

Combining High-Pressure NMR and Geometrical Sampling to Obtain a Full Topological Description of Protein Folding Landscapes: Application to the Folding of Two MAX Effectors from Magnaporthe oryzae

by Cécile Dubois, Mounia Lahfa, Joana Pissarra, Karine de Guillen, Philippe Barthe, Thomas Kroj, Christian Roumestand and André Padilla

Int. J. Mol. Sci. 2022, 23(10), 5461; https://doi.org/10.3390/ijms23105461 - 13 May 2022

Cited by 2 | Viewed by 1568

Abstract

Despite advances in experimental and computational methods, the mechanisms by which an unstructured polypeptide chain regains its unique three-dimensional structure remains one of the main puzzling questions in biology. Single-molecule techniques, ultra-fast perturbation and detection approaches and improvement in all-atom and coarse-grained simulation methods have greatly deepened our understanding of protein folding and the effects of environmental factors on folding landscape. However, a major challenge remains the detailed characterization of the protein folding landscape. Here, we used high hydrostatic pressure 2D NMR spectroscopy to obtain high-resolution experimental structural information in a site-specific manner across the polypeptide sequence and along the folding reaction coordinate. We used this residue-specific information to constrain Cyana3 calculations, in order to obtain a topological description of the entire folding landscape. This approach was used to describe the conformers populating the folding landscape of two small globular proteins, AVR-Pia and AVR-Pib, that belong to the structurally conserved but sequence-unrelated MAX effectors superfamily. Comparing the two folding landscapes, we found that, in spite of their divergent sequences, the folding pathway of these two proteins involves a similar, inescapable, folding intermediate, even if, statistically, the routes used are different. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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14 pages, 2513 KiB

Open AccessArticle

Probing and Manipulating the Lateral Pressure Profile in Lipid Bilayers Using Membrane-Active Peptides—A Solid-State ¹⁹F NMR Study

by Stephan L. Grage, Sergii Afonin, Marco Ieronimo, Marina Berditsch, Parvesh Wadhwani and Anne S. Ulrich

Int. J. Mol. Sci. 2022, 23(9), 4544; https://doi.org/10.3390/ijms23094544 - 20 Apr 2022

Cited by 2 | Viewed by 1675

Abstract

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A ¹⁹F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state ¹⁹F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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Review

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33 pages, 21692 KiB

Open AccessReview

Three Decades of REDOR in Protein Science: A Solid-State NMR Technique for Distance Measurement and Spectral Editing

by Orsolya Toke

Int. J. Mol. Sci. 2023, 24(17), 13637; https://doi.org/10.3390/ijms241713637 - 4 Sep 2023

Cited by 1 | Viewed by 1222

Abstract

Solid-state NMR (ss-NMR) is a powerful tool to investigate noncrystallizable, poorly soluble molecular systems, such as membrane proteins, amyloids, and cell walls, in environments that closely resemble their physical sites of action. Rotational-echo double resonance (REDOR) is an ss-NMR methodology, which by reintroducing heteronuclear dipolar coupling under magic angle spinning conditions provides intramolecular and intermolecular distance restraints at the atomic level. In addition, REDOR can be exploited as a selection tool to filter spectra based on dipolar couplings. Used extensively as a spectroscopic ruler between isolated spins in site-specifically labeled systems and more recently as a building block in multidimensional ss-NMR pulse sequences allowing the simultaneous measurement of multiple distances, REDOR yields atomic-scale information on the structure and interaction of proteins. By extending REDOR to the determination of ¹H–X dipolar couplings in recent years, the limit of measurable distances has reached ~15–20 Å, making it an attractive method of choice for the study of complex biomolecular assemblies. Following a methodological introduction including the most recent implementations, examples are discussed to illustrate the versatility of REDOR in the study of biological systems. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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22 pages, 5480 KiB

Open AccessReview

Recent Advances in NMR Protein Structure Prediction with ROSETTA

by Julia Koehler Leman and Georg Künze

Int. J. Mol. Sci. 2023, 24(9), 7835; https://doi.org/10.3390/ijms24097835 - 25 Apr 2023

Cited by 8 | Viewed by 3116

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture. Computational modeling techniques can complement sparse NMR datasets (<1 restraint per residue) with additional structural information to elucidate protein structures in these difficult cases. The Rosetta software for protein structure modeling and design is used by structural biologists for structure determination tasks in which limited experimental data is available. This review gives an overview of the computational protocols available in the Rosetta framework for modeling protein structures from NMR data. We explain the computational algorithms used for the integration of different NMR data types in Rosetta. We also highlight new developments, including modeling tools for data from paramagnetic NMR and hydrogen–deuterium exchange, as well as chemical shifts in CS-Rosetta. Furthermore, strategies are discussed to complement and improve structure predictions made by the current state-of-the-art AlphaFold2 program using NMR-guided Rosetta modeling. Full article

(This article belongs to the Special Issue NMR Tools and Their Application for Structural Biology of Bioactive Proteins)

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