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Visualizing a protein's molecular motions has been a long standing topic of research in the biophysics community. Largely this has been done by exploiting nuclear magnetic resonance spectroscopy (NMR), and arguably no protein's molecular motions have been better characterized by NMR than that of ubiquitin (Ub), a 76 amino acid polypeptide essential in ubiquitination-a key regulatory system within cells. Herein, we discuss ubiquitin's conformational plasticity as visualized, at atomic resolution, by more than 35 years of NMR work. In our discussions we point out the differences between data acquired in vitro, ex vivo, as well as in vivo and stress the need to investigate Ub's conformational plasticity in more biologically representative backgrounds., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Sensitivity enhanced dynamic nuclear polarization solid-state NMR is emerging as a powerful technique for probing the structural properties of conformationally homogenous and heterogenous biomolecular species irrespective of size at atomic resolution within their native environments. Herein we detail advancements that have made acquiring such data, specifically within the confines of intact bacterial and eukaryotic cell a reality and further discuss the type of structural information that can presently be garnered by the technique's exploitation. Subsequently, we discuss bottlenecks that have thus far curbed cellular DNP-ssNMR's broader adoption namely due a lack of sensitivity and spectral resolution. We also explore possible solutions ranging from utilization of new pulse sequences, design of better performing polarizing agents, and application of additional biochemical/ cell biological methodologies., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod-Wyman-Changeux model., Competing Interests: Competing interests statement:The authors declare no competing interest.

Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [ 13 C, 15 N] labeled ubiquitin. We report that SNAPol-1 passively diffuses and homogenously distributes within whole cells and cell nuclei providing ubiquitin spectra of high sensitivity and remarkably improved spectral resolution. For cell nuclei, physical enrichment facilitates a further 4-fold decrease in measurement time and provides an exclusive structural view of the nuclear ubiquitin pool. Taken together, these advancements enable atomic interrogation of protein conformational plasticity at atomic resolution and with sub-cellular specificity., Competing Interests: The authors have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional 13 C/ 15 N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton ( 1 H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3-5 days.

Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ~ 200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation. Since nucleosomes are known to self-associate, these dense phases may induce extensive interactions between nucleosomes, which could interfere with protein-binding studies. Here, we characterized the packing of nucleosomes in the dense phase created by sedimentation using NMR and small-angle X-ray scattering (SAXS) experiments. We found that nucleosome sediments are gels with variable degrees of solidity, have nucleosome concentration close to that found in crystals, and are stable for weeks under high-speed magic angle spinning (MAS). Furthermore, SAXS data recorded on recovered sediments indicate that there is no pronounced long-range ordering of nucleosomes in the sediment. Finally, we show that the sedimentation approach can also be used to study low-affinity protein interactions with the nucleosome. Together, our results give new insights into the sample characteristics of nucleosome sediments for ssNMR studies and illustrate the broad applicability of sedimentation-based NMR studies., Competing Interests: Competing interests. The authors declare that they have no conflict of interest.

Solution-state NMR spectroscopy has become a powerful tool to study soluble proteins in cells, provided that they tumble sufficiently fast. In addition, cryo-electron tomography (cryo-ET) has recently displayed a tremendous potential to probe structures of large proteins and assemblies in their native cellular environments. However, challenges remain to obtain atomic-level information in native cell settings for proteins that are small, disordered, or are strongly engaged in intermolecular interactions. In this Minireview, we discuss recent progress in using sensitivity enhanced solid-state NMR spectroscopy methods in the context of cellular structural biology., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid-state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high-sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning. Our results pave the way for structural studies of larger proteins or protein complexes inside human cells, which have remained elusive to in-cell solution-state NMR spectroscopy due to molecular size limitations., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Quantitative evaluation of binding affinity changes upon mutations is crucial for protein engineering and drug design. Machine learning-based methods are gaining increasing momentum in this field. Due to the limited number of experimental data, using a small number of sensitive predictive features is vital to the generalization and robustness of such machine learning methods. Here we introduce a fast and reliable predictor of binding affinity changes upon single point mutation, based on a random forest approach. Our method, iSEE, uses a limited number of interface Structure, Evolution, and Energy-based features for the prediction. iSEE achieves, using only 31 features, a high prediction performance with a Pearson correlation coefficient (PCC) of 0.80 and a root mean square error of 1.41 kcal/mol on a diverse training dataset consisting of 1102 mutations in 57 protein-protein complexes. It competes with existing state-of-the-art methods on two blind test datasets. Predictions for a new dataset of 487 mutations in 56 protein complexes from the recently published SKEMPI 2.0 database reveals that none of the current methods perform well (PCC < 0.42), although their combination does improve the predictions. Feature analysis for iSEE underlines the significance of evolutionary conservations for quantitative prediction of mutation effects. As an application example, we perform a full mutation scanning of the interface residues in the MDM2-p53 complex., (© 2018 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores., Competing Interests: The authors declare no conflict of interest.

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA-binding modules such as IF2-C2, IF1 and subdomains II of elongation factors EF-Tu and EF-G. Structural comparisons have resulted in a model that describes the interaction between IF2-G3 and the 30S ribosomal subunit., (© 2016 The Protein Society.)

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization-based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

Solid-state NMR spectroscopy (ssNMR) provides increasing possibilities to examine membrane proteins in different molecular settings, ranging from synthetic bilayers to whole cells. This flexibility often enables ssNMR experiments to be directly correlated with membrane protein function. In this contribution, we discuss experimental aspects of such studies starting with protein expression and labeling, leading to membrane protein isolation or to membrane proteins in a cellular environment. We show that optimized procedures can depend on aspects such as the achieved levels of expression, the stability of the protein during purification or proper refolding. Dealing with native membrane samples, such as isolated cellular membranes, can alleviate or entirely remove such biochemical challenges. Subsequently, we outline ssNMR experiments that involve the use of magic-angle-spinning and can be used to study membrane protein structure and their functional aspects. We pay specific attention to spectroscopic issues such as sensitivity and spectral resolution. The latter aspect can be controlled using a combination of tailored preparation procedures with solid-state NMR experiments that simplify the spectral analysis using specific filtering and correlation methods. Such approaches have already provided access to obtain structural views of membrane proteins and study their function in lipid bilayers. Ongoing developments in sample preparation and NMR methodology, in particular in using hyperpolarization or proton-detection schemes, offer additional opportunities to study membrane proteins close to their cellular function. These considerations suggest a further increase in the potential of using solid-state NMR in the context of prokaryotic or eukaryotic membrane protein systems in the near future., (© 2015 Elsevier Inc. All rights reserved.)