Structural Studies Of Proteins Research Articles

Optimization of 15N–13C double-resonance NMR experiments under low temperature magic angle spinning dynamic nuclear polarization conditions

Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR carried out at 25 K enables rapid acquisition of multi-dimensional 13C–15N correlation spectra for protein structure studies and resonance assignment. Under commonly used DNP conditions, solvent deuteration reduces 1H–15N cross polarization (CP) efficiencies, necessitates more careful optimization, and requires longer high-power 15N radio-frequency pulses. The sensitivity of 2D heteronuclear correlation experiments is potentially impaired. Here we show that 2D 15N-13C experiments based on 13C-15N transferred echo double resonance (TEDOR) methods outperform 2D experiments based on CP transfers in a fully deuterated solvent, and are competitive with CP-based experiments when the solvent is only partially deuterated. Additionally, we show that optimization of TEDOR-based 2D experiments is simpler than optimization of CP-based experiments under 25 K MAS conditions.

Precision defect integrated graphene as reliable support membrane for high-resolution cryo-transmission electron microscopy

Graphene is an excellent support film for high-resolution transmission electron microscopy (TEM) but its use with biological samples, notably in cryo-TEM, is hindered by its inherent hydrophobicity. Whereas surface treatments have been proposed to render graphene hydrophilic, they are often difficult to reproduce due to a lack of information on the structural changes that modify the wetting properties of graphene. This study aims to correlate the atomic structure of graphene with its wetting properties to allow a reproducible protocol to advance its application in cryo-TEM. We follow the change in the atomic structure of graphene as a function of low-energy hydrogen plasma treatment duration on monolayer graphene transferred onto TEM grids. With finely controlled plasma exposure, partial hydrogenation, monoatomic vacancies, and pores of a few nanometers are realized in the graphene. The introduction of defects (vacancies and pores) enables the formation of continuous layers of vitreous ice on TEM grids. Grids with defect-integrated graphene are reproduced and used in the vitrification of the mouse serotonin 5-HT3 receptor, a membrane protein. Single particle analysis of the membrane protein on graphene compared to conventional holey carbon film give insight into the strengths and discretions in using graphene membrane for protein structural studies.

Post-translational modifications in the Protein Data Bank.

Proteins frequently undergo covalent modification at the post-translational level, which involves the covalent attachment of chemical groups onto amino acids. This can entail the singular or multiple addition of small groups, such as phosphorylation; long-chain modifications, such as glycosylation; small proteins, such as ubiquitination; as well as the interconversion of chemical groups, such as the formation of pyroglutamic acid. These post-translational modifications (PTMs) are essential for the normal functioning of cells, as they can alter the physicochemical properties of amino acids and therefore influence enzymatic activity, protein localization, protein-protein interactions and protein stability. Despite their inherent importance, accurately depicting PTMs in experimental studies of protein structures often poses a challenge. This review highlights the role of PTMs in protein structures, as well as the prevalence of PTMs in the Protein Data Bank, directing the reader to accurately built examples suitable for use as a modelling reference.

Fibrinogen post-translational modifications are biochemical determinants of fibrin clot properties and interactions.

The structure of fibrinogen and resulting fibrin formed during the coagulation process have important biological functions in human physiology and pathology. Fibrinogen post-translational modifications (PTMs) increase the complexity of the protein structure and many studies have emphasized thepotential associations of post-translationally altered fibrinogen with the formation of a fibrin clot with a prothrombotic phenotype. However, the mechanisms by which PTMs exert their action on fibrinogen, and their causal association with disease pathogenesis are relatively unexplored. Moreover, the significance of fibrinogen PTMs in health has yet to be appreciated. In this review, the impact of fibrinogen PTMs on fibrinogen functionality is discussed from a biochemical perspective, emphasizing the potential mechanisms by which PTMs mediate the acquisition of altered fibrinogen properties. A brief discussion on dysfibrinogenemias of genetic origin, attributed to single point variations of the fibrinogen molecule is also provided, highlighting the influence that amino acid properties have on fibrinogen structure, properties, and molecular interactions that arise during thrombus formation.

Towards cost-effective side-chain isotope labelling of proteins expressed in human cells.

Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.

Exploring serial crystallography for drug discovery.

Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today - conventional X-ray crystallography - is that it only provides structural information about the protein complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room temperature (RT). Here, we explore the use of serial crystallography to determine the structures of the pharmaceutical target, soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of RT ligand-bound structures of our target protein. From a comparison between the RT structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in the ligand-binding mode and observe that flexible loops are better resolved at RT. Finally, we discuss the current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.

Exploring genetic codon expansion for unnatural amino acid incorporation in filamentous fungus Aspergillus nidulans

Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyrCUA (Ec. tRNATyrCUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans, they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O-methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function.

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry.

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics.

A novel variant in the FLNB gene associated with spondylocarpotarsal synostosis syndrome.

Genetic disorders involved in skeleton system arise due to the disturbance in skeletal development, growth and homeostasis. Filamin B is an actin binding protein which is large dimeric protein which cross link actin cytoskeleton filaments into dynamic structure. A single nucleotide changes in the FLNB gene causes spondylocarpotarsal synostosis syndrome, a rare bone disorder due to which the fusion of carpels and tarsals synostosis occurred along with fused vertebrae. In the current study we investigated a family residing in north-western areas of Pakistan. The whole exome sequencing of proband was performed followed by Sanger sequencing of all family members of the subject to validate the variant segregation within the family. Bioinformatics tools were utilized to assess the pathogenicity of the variant. Whole Exome Sequencing revealed a novel variant (NM_001457: c.209C>T and p.Pro70Leu) in the FLNB gene which was homozygous missense mutation in the FLNB gene. The variant was further validated and visualized by Sanger sequencing and protein structure studies respectively as mentioned before. The findings have highlighted the importance of the molecular diagnosis in SCT (spondylocarpotarsal synostosis syndrome) for genetic risk counselling in consanguineous families.

Antibodies expand the scope of angiotensin receptor pharmacology.

G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

Proteins Can Withstand More Extensive Labeling while Providing Accurate Structural Information in Covalent Labeling-Mass Spectrometry.

Diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) has been extensively utilized to study protein structure and interactions owing to its ease of use, commercial availability, and broad labeling of nucleophilic residues. During typical CL-MS experiments with DEPC, the extent of labeling is kept low to avoid any structural perturbations resulting from covalent modification of the protein. In this study, we demonstrate that proteins can be labeled more extensively via DEPC and still provide accurate structural information. To show this, we modeled labeling kinetics over a range of DEPC concentrations and used molecular dynamics simulations to investigate the molecular-level effects of extensive labeling on the protein structure. Our results indicate that higher extents of DEPC labeling do not significantly perturb the protein structure and can lead to improved precision, detectability of labeled peptides, and protein structural resolution. Furthermore, higher extents of labeling enable better identification of protein-ligand binding sites where lower extents of modification provide ambiguous results.

Methods for research on structures and functions of traditional Chinese medicine proteins

Traditional Chinese medicine proteins(TCMPs) not only have nutritional values and biological activities but also serve as key enzymes in the synthesis of pharmacodynamic components in traditional Chinese medicines. They play a role in the synthesis of pharmacodynamic components by regulating biosynthesis and selective synthesis pathways and controlling drug quality and stability. The recent years have witnessed great progress in the research on the structures and functions of proteins using various methods and technologies. However, the research on the structures and functions of TCMPs lags behind. Therefore, it is urgent to study the structures and functions of TCMPs using modern means to promote the discovery of innovative drugs based on TCMPs and clarify the synthesis pathways of pharmacodynamic components. This study introduces the latest techniques for studying protein structures and functions, including spectroscopy, mass spectrometry, nuclear magnetic resonance, X-ray crystal diffraction, microscopy, and structure prediction. Furthermore, this paper introduces the methods for protein functional studies, including liquid chromatography-mass spectrometry, co-immunoprecipitation, yeast two-hybrid, and pull-down assay. By systematically reviewing these techniques and methods, this paper provides technical references for the structural identification and functional studies of TCMPs, with the aim of promoting the in-depth exploration of the structures and functions of TCMPs.

An analysis of double-quantum coherence ESR in an N-spin system: Analytical expressions and predictions.

Electron spin resonance pulsed dipolar spectroscopy (PDS) has become popular in protein 3D structure analysis. PDS studies yield distance distributions between a pair or multiple pairs of spin probes attached to protein molecules, which can be used directly in structural studies or as constraints in theoretical predictions. Double-quantum coherence (DQC) is a highly sensitive and accurate PDS technique to study protein structures in the solid state and under physiologically relevant conditions. In this work, we have derived analytical expressions for the DQC signal for a system with N-dipolar coupled spin-1/2 particles in the solid state. The expressions are integrated over the relevant spatial parameters to obtain closed form DQC signal expressions. These expressions contain the concentration-dependent "instantaneous diffusion" and the background signal. For micromolar and lower concentrations, these effects are negligible. An approximate analysis is provided for cases of finite pulses. The expressions obtained in this work should improve the analysis of DQC experimental data significantly, and the analytical approach could be extended easily to a wide range of magnetic resonance phenomena.

HydraProt: A New Deep Learning Tool for Fast and Accurate Prediction of Water Molecule Positions for Protein Structures.

Water molecules are integral to the structural stability of proteins and vital for facilitating molecular interactions. However, accurately predicting their precise position around protein structures remains a significant challenge, making it a vibrant research area. In this paper, we introduce HydraProt (deep Hydration of Proteins), a novel methodology for predicting precise positions of water molecule oxygen atoms around protein structures, leveraging two interconnected deep learning architectures: a 3D U-net and a Multi-Layer Perceptron (MLP). Our approach starts by introducing a coarse voxel-based representation of the protein, which allows for rapid sampling of candidate water positions via the 3D U-net. These water positions are then assessed by embedding the water-protein relationship in the Euclidean space by means of an MLP. Finally, a postprocessing step is applied to further refine the MLP predictions. HydraProt surpasses existing state-of-the-art approaches in terms of precision and recall and has been validated on large data sets of protein structures. Notably, our method offers rapid inference runtime and should constitute the method of choice for protein structure studies and drug discovery applications. Our pretrained models, data, and the source code required to reproduce these results are accessible at https://github.com/azamanos/HydraProt.

Multilevel superposition for deciphering the conformational variability of protein ensembles.

The dynamics and variability of protein conformations are directly linked to their functions. Many comparative studies of X-ray protein structures have been conducted to elucidate the relevant conformational changes, dynamics and heterogeneity. The rapid increase in the number of experimentally determined structures has made comparison an effective tool for investigating protein structures. For example, it is now possible to compare structural ensembles formed by enzyme species, variants or the type of ligands bound to them. In this study, the author developed a multilevel model for estimating two covariance matrices that represent inter- and intra-ensemble variability in the Cartesian coordinate space. Principal component analysis using the two estimated covariance matrices identified the inter-/intra-enzyme variabilities, which seemed to be important for the enzyme functions, with the illustrative examples of cytochrome P450 family 2 enzymes and class A $\beta$-lactamases. In P450, in which each enzyme has its own active site of a distinct size, an active-site motion shared universally between the enzymes was captured as the first principal mode of the intra-enzyme covariance matrix. In this case, the method was useful for understanding the conformational variability after adjusting for the differences between enzyme sizes. The developed method is advantageous in small ensemble-size problems and hence promising for use in comparative studies on experimentally determined structures where ensemble sizes are smaller than those generated, for example, by molecular dynamics simulations.

A nanocomposite film with layer-by-layer self-assembled gold nanospheres driven by cucurbit[7]uril for the selective transport of l-tryptophan and lysozyme

Selective separation of amino acids and proteins is crucial in various areas of research, including proteomics, protein structure and function studies, protein purification and drug development, and biosensing and biodetection. A nanocomposite film is formed by combining layer-by-layer self-assembled gold nanospheres (AuNPs) driven by cucurbit[7]uril (CB[7]) and polymethyl methacrylate (PMMA) film. Due to the host-guest interactions, the selective transmission of l-tryptophan in the nanocomposite film is confirmed by the current-voltage measurements using a picoammeter. Furthermore, by adjusting the particle size of AuNPs to increase channel size, lysozyme containing multiple tryptophan residues can selectively pass through the nanocomposite film, indicating the high versatility and adaptability of the nanocomposite film. This study will provide a new direction for the selective separation of amino acids and proteins.

Proteomics and Its Current Application in Biomedical Area: Concise Review.

Biomedical researchers tirelessly seek cutting-edge technologies to advance disease diagnosis, drug discovery, and therapeutic interventions, all aimed at enhancing human and animal well-being. Within this realm, proteomics stands out as a pivotal technology, focusing on extensive studies of protein composition, structure, function, and interactions. Proteomics, with its subdivisions of expression, structural, and functional proteomics, plays a crucial role in unraveling the complexities of biological systems. Various sophisticated techniques are employed in proteomics, including polyacrylamide gel electrophoresis, mass spectrometry analysis, NMR spectroscopy, protein microarray, X-ray crystallography, and Edman sequencing. These methods collectively contribute to the comprehensive understanding of proteins and their roles in health and disease. In the biomedical field, proteomics finds widespread application in cancer research and diagnosis, stem cell studies, and the diagnosis and research of both infectious and noninfectious diseases. In addition, it plays a pivotal role in drug discovery and the emerging frontier of personalized medicine. The versatility of proteomics allows researchers to delve into the intricacies of molecular mechanisms, paving the way for innovative therapeutic approaches. As infectious and noninfectious diseases continue to emerge and the field of biomedical research expands, the significance of proteomics becomes increasingly evident. Keeping abreast of the latest developments in proteomics applications becomes paramount for the development of therapeutics, translational research, and study of diverse diseases. This review aims to provide a comprehensive overview of proteomics, offering a concise outline of its current applications in the biomedical domain. By doing so, it seeks to contribute to the understanding and advancement of proteomics, emphasizing its pivotal role in shaping the future of biomedical research and therapeutic interventions.

Controlling the incorporation of fluorinated amino acids in human cells and its structural impact.

Fluorinated aromatic amino acids (FAAs) are promising tools when studying protein structure and dynamics by NMR spectroscopy. The incorporation FAAs in mammalian expression systems has been introduced only recently. Here, we investigate the effects of FAAs incorporation in proteins expressed in human cells, focusing on the probability of incorporation and its consequences on the 19 F NMR spectra. By combining 19 F NMR, direct MS and x-ray crystallography, we demonstrate that the probability of FAA incorporation is only a function of the FAA concentration in the expression medium and is a pure stochastic phenomenon. In contrast with the MS data, the x-ray structures of carbonic anhydrase II reveal that while the 3D structure is not affected, certain positions lack fluorine, suggesting that crystallization selectively excludes protein molecules featuring subtle conformational modifications. This study offers a predictive model of the FAA incorporation efficiency and provides a framework for controlling protein fluorination in mammalian expression systems.

Progress, Challenges and Opportunities of NMR and XL-MS for Cellular Structural Biology.

The validity of protein structures and interactions, whether determined under ideal laboratory conditions or predicted by AI tools such as Alphafold2, to precisely reflect those found in living cells remains to be examined. Moreover, understanding the changes in protein structures and interactions in response to stimuli within living cells, under both normal and disease conditions, is key to grasping proteins' functionality and cellular processes. Nevertheless, achieving high-resolution identification of these protein structures and interactions within living cells presents a technical challenge. In this Perspective, we summarize the recent advancements in in-cell nuclear magnetic resonance (NMR) and in vivo cross-linking mass spectrometry (XL-MS) for studying protein structures and interactions within a cellular context. Additionally, we discuss the challenges, opportunities, and potential benefits of integrating in-cell NMR and in vivo XL-MS in future research to offer an exhaustive approach to studying proteins in their natural habitat.