Large Protein Assemblies Research Articles

Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Size-Exclusion Chromatography for Analysis of Complex Protein Samples.

The growing use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for studying membrane proteins, large protein assemblies, and highly disulfide-bonded species is often challenged by the presence in the sample of large amounts of lipids, protein ligands, and/or highly ionizable reducing agents. Here, we describe how a short size-exclusion chromatography (SEC) column can be integrated with a conventional temperature-controlled HDX-MS setup to achieve fast and online removal of unwanted species from the HDX sample prior to chromatographic separation and MS analysis. Dual-mode valves permit labeled proteins eluting after SEC to be directed to the proteolytic and chromatographic columns, while unwanted sample components are led to waste. The SEC-coupled HDX-MS method allows analyses to be completed with lower or similar back-exchange compared to conventional experiments. We demonstrate the suitability of the method for the analysis of challenging protein samples, achieving efficient online removal of lipid components from protein-lipid systems, depletion of an antibody from an antigen during epitope mapping, and elimination of MS interfering compounds such as tris(2-carboxyethyl)phosphine (TCEP) during HDX-MS analysis of a disulfide-bonded protein. The implementation of the short SEC column to the conventional HDX-MS setup is straightforward and could be of significant general utility during the HDX-MS analysis of complex protein states.

Protonation-Deprotonation Switch Controls the Amyloid-like Misfolding of Nucleic-Acid-Binding Domains of TDP-43.

Nutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43tRRM) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43tRRM forms a misfolded amyloid-like protein assembly, β form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-β transition. We observed that D105A and H256Q resembled TDP-43tRRM in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the β form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43tRRM and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.

Protein interaction patterns in Arabidopsis thaliana leaf mitochondria change in dependence to light

Mitochondrial biology is underpinned by the presence and activity of large protein assemblies participating in the organelle-located steps of respiration, TCA-cycle, glycine oxidation, and oxidative phosphorylation. While the enzymatic roles of these complexes are undisputed, little is known about the interactions of the subunits beyond their presence in these protein complexes and their functions in regulating mitochondrial metabolism. By applying one of the most important regulatory cues for plant metabolism, the presence or absence of light, we here assess changes in the composition and molecular mass of protein assemblies involved in NADH-production in the mitochondrial matrix and in oxidative phosphorylation by employing a differential complexome profiling strategy. Covering a mass up to 25 MDa, we demonstrate dynamic associations of matrix enzymes and of components involved in oxidative phosphorylation. The data presented here form the basis for future studies aiming to advance our understanding of the role of protein:protein interactions in regulating plant mitochondrial functions.

Paramagnetic Solid-State NMR to Localize the Metal-Ion Cofactor in an Oligomeric DnaB Helicase.

Paramagnetic metal ions can be inserted into ATP‐fueled motor proteins by exchanging the diamagnetic Mg2+ cofactor with Mn2+ or Co2+. Then, paramagnetic relaxation enhancement (PRE) or pseudo‐contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition‐state ATP‐analogue ADP:AlF4 − and single‐stranded DNA using solid‐state NMR and a structure‐calculation protocol employing CYANA. We discuss and compare the use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies. 31P PCSs induced in the Co2+‐containing sample are then used to localize the DNA phosphate groups on the Co2+ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.

Isolating and Analyzing Protein Containing Granules from Cells

Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: SEC separation of cross-linked mammalian cell lysates Alternate Protocol: Preparation of non-crosslinked mammalian cells Basic Protocol 2: SEC separation of E. coli lysate Support Protocol 1: Detecting protein of interest by ELISA Support Protocol 2: TCA precipitation of SEC fractions.

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line.

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.

High-Pressure Electrospray Ionization Yields Supercharged Protein Complexes from Native Solutions While Preserving Noncovalent Interactions.

Increasing charge state of protein complexes from native solutions while preserving noncovalent interactions in native mass spectrometry (MS) offers great opportunity to gain deeper insights into gas-phase protein structures. Several previous studies have disclosed the possibility of high pressure in supercharging small proteins, whereas its capability to supercharge large protein assemblies under native conditions and how it might affect protein structures remain open questions. Herein, we demonstrated that the high-pressure-induced supercharging strategy affords unique advantages of supercharging protein complexes with the highest charge state surpassing the Rayleigh limit (ZR) and concurrently preserving native-like topology. By examining 32 proteins and protein complexes with molecular weights (MWs) ranging from 8.58 to 801 kDa, we demonstrated that the increased average charge states of macromolecular ions have a strong dependence on the surface areas of native protein conformations and MWs. Factors that might contribute to the high-pressure-induced supercharging capability toward macromolecular ions were discussed. Furthermore, using collision cross section (CCS) variation as a function of charge state, we investigate the effects of gas pressure and charge states on gas-phase structures of proteins and protein complexes. Smaller proteins have the largest CCS variations once supercharged, while macromolecular protein complexes are less affected. The results revealed that both surface density of charge and charged surface basic residues contribute to the observed CCS-charge disciplines for all the macromolecules investigated. Taken together, the results presented here indicate that increasing gas pressure in the ion source affords a rapid, simple, and controlled supercharging method, offering the potency of facilitating further applications of native top-down MS analysis with improved transmission, fragmentation, and detection efficiency.

First person – Mang Zhu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Mang Zhu is first author on ‘Proteomic analysis reveals the direct recruitment of intrinsically disordered regions to stress granules in S. cerevisiae’, published in JCS. Mang is a PhD candidate in the lab of Thibault Mayor at Michael Smith Laboratories, Vancouver, Canada, investigating how large protein assemblies are formed in cells.

SWI/SNF complex-deficient soft tissue neoplasms: An update.

The SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex is a large multi-subunit protein assembly that orchestrates chromatin compaction and accessibility for gene transcription in an ATP-dependent manner. As a key epigenetic regulator, the SWI/SNF complex coordinates gene expression, cell proliferation and differentiation, and its biologic functions, in part, antagonize the polycomb repressive complex 2. The mammalian SWI/SNF complex consists of 15 subunits encoded by 29 genes, some of which are recurrently mutated in human cancers, in the germline or sporadic setting. Most SWI/SNF-deficient tumors share common "rhabdoid" cytomorphology. SMARCB1 (INI1) is the subunit most frequently inactivated in soft tissue neoplasms. Specifically, SMARCB1 deficiency is observed as the genetic hallmark in virtually all malignant rhabdoid tumors, and most cases of epithelioid sarcoma and poorly differentiated chordoma. In addition, subsets of myoepithelial carcinoma (10-40%), extraskeletal myxoid chondrosarcoma (20%), epithelioid schwannoma (40%), and epithelioid malignant peripheral nerve sheath tumor (70%) demonstrate SMARCB1 loss. The gene encoding the SS18 subunit is involved in the SS18-SSX rearrangement, which is pathognomonic of synovial sarcoma and indirectly inactivates SMARCB1. Finally, undifferentiated SMARCA4-deficient thoracic sarcomas are defined by SMARCA4 subunit inactivation, leading to SMARCA4 and SMARCA2 loss. Rarely, inactivation of alternate but biologically equivalent key regulators can substitute for canonical subunit deficiency, such as SMARCA4 inactivation in cases of SMARCB1-retained epithelioid sarcoma. This review briefly highlights SWI/SNF complex biologic functions and its roles in human cancer and provides a detailed update on recent advances in soft tissue neoplasms with canonical SWI/SNF complex deficiency, correlating morphologic, genomic, and immunohistochemical findings.

A homozygous MRPL24 mutation causes a complex movement disorder and affects the mitoribosome assembly

Mitochondrial ribosomal protein large 24 (MRPL24) is 1 of the 82 protein components of mitochondrial ribosomes, playing an essential role in the mitochondrial translation process.We report here on a baby girl with cerebellar atrophy, choreoathetosis of limbs and face, intellectual disability and a combined defect of complexes I and IV in muscle biopsy, caused by a homozygous missense mutation identified in MRPL24. The variant predicts a Leu91Pro substitution at an evolutionarily conserved site. Using human mutant cells and the zebrafish model, we demonstrated the pathological role of the identified variant. In fact, in fibroblasts we observed a significant reduction of MRPL24 protein and of mitochondrial respiratory chain complex I and IV subunits, as well a markedly reduced synthesis of the mtDNA-encoded peptides. In zebrafish we demonstrated that the orthologue gene is expressed in metabolically active tissues, and that gene knockdown induced locomotion impairment, structural defects and low ATP production. The motor phenotype was complemented by human WT but not mutant cRNA. Moreover, sucrose density gradient fractionation showed perturbed assembly of large subunit mitoribosomal proteins, suggesting that the mutation leads to a conformational change in MRPL24, which is expected to cause an aberrant interaction of the protein with other components of the 39S mitoribosomal subunit.

An expanded library of orthogonal split inteins enables modular multi-peptide assemblies

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.

Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers.

Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.

Nucleoporin 160 Regulates Flowering through Anchoring HOS1 for Destabilizing CO in Arabidopsis

Nuclear pore complexes (NPCs), which comprise multiple copies of nucleoporins (Nups), are large protein assemblies embedded in the nuclear envelope connecting the nucleus and cytoplasm. Although it has been known that Nups affect flowering in Arabidopsis, the underlying mechanisms are poorly understood. Here, we show that loss of function of Nucleoporin 160 (Nup160) leads to increased abundance of CONSTANS (CO) protein and the resulting upregulation of FLOWERING LOCUS T (FT) specifically in the morning. We demonstrate that Nup160 regulates CO protein stability through affecting NPC localization of an E3-ubiquitin ligase, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1), which destabilizes CO protein in the morning period. Taken together, these results provide a mechanistic understanding of Nup function in the transition from vegetative to reproductive growth, suggesting that deposition of HOS1 at NPCs by Nup160 is essential for preventing precocious flowering in response to photoperiod in Arabidopsis.

Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting.

The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose-response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies.

Chemical Cross-Linking and Mass Spectrometric Analysis of the Endogenous Yeast Exosome Complexes.

Chemical cross-linking and mass spectrometric readout (CX-MS) has become a useful toolkit for structural analysis of protein complexes. CX-MS enables rapid detection of a larger number of cross-link peptides from the chemically cross-linked protein assembly, providing invaluable cross-link spatial restraints to understand the architecture of the complex. Since CX-MS is complementary with other structural and computational modeling tools, it can be used for integrative structural determination of large native protein assemblies. However, due to technical limitations, current CX-MS applications have still been predominantly confined to complexes reconstituted from recombinant proteins where large amount of purified materials are available. Cross-linking and hybrid structural proteomic analysis of endogenous protein complexes remains a challenge. In this chapter, we present a protocol that efficiently couples affinity capture of endogenous complexes with sensitive CX-MS analysis, with particular application to the yeast RNA processing exosome complexes.

Quantitative Structural Interpretation of Protein Crosslinks

Chemical crosslinking, combined with mass spectrometry analysis, is a key source of information for characterizing the structure of large protein assemblies, in the context of molecular modeling. In most approaches, the interpretation is limited to simple spatial restraints, neglecting physico-chemical interactions between the crosslinker and the protein and their flexibility. Here we present a method, named NRGXL (new realistic grid for crosslinks), which models the flexibility of the crosslinker and the linked side-chains, by explicitly sampling many conformations. Also, the method can efficiently deal with overall protein dynamics. This method creates a physical model of the crosslinker and associated energy. A classifier based on it outperforms others, based on Euclidean distance or solvent-accessible distance and its efficiency makes it usable for validating 3D models from crosslinking data. NRGXL is freely available as a web server at: https://nrgxl.pasteur.fr.

Germline Maintenance Through the Multifaceted Activities of GLH/Vasa in Caenorhabditis elegans P Granules.

Vasa homologs are ATP-dependent DEAD-box helicases, multipotency factors, and critical components that specify and protect the germline. They regulate translation, amplify piwi-interacting RNAs (piRNAs), and act as RNA solvents; however, the limited availability of mutagenesis-derived alleles and their wide range of phenotypes have complicated their analysis. Now, with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), these limitations can be mitigated to determine why protein domains have been lost or retained throughout evolution. Here, we define the functional motifs of GLH-1/Vasa in Caenorhabditis elegans using 28 endogenous, mutant alleles. We show that GLH-1's helicase activity is required to retain its association with P granules. GLH-1 remains in P granules when changes are made outside of the helicase and flanking domains, but fertility is still compromised. Removal of the glycine-rich repeats from GLH proteins progressively diminishes P-granule wetting-like interactions at the nuclear periphery. Mass spectrometry of GLH-1-associated proteins implies conservation of a transient piRNA-amplifying complex, and reveals a novel affinity between GLH-1 and three structurally conserved PCI (26S Proteasome Lid, COP9, and eIF3) complexes or "zomes," along with a reciprocal aversion for assembled ribosomes and the 26S proteasome. These results suggest that P granules compartmentalize the cytoplasm to exclude large protein assemblies, effectively shielding associated transcripts from translation and associated proteins from turnover. Within germ granules, Vasa homologs may act as solvents, ensuring mRNA accessibility by small RNA surveillance and amplification pathways, and facilitating mRNA export through germ granules to initiate translation.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

Dynamic graphical models of molecular kinetics

Most current molecular dynamics simulation and analysis methods rely on the idea that the molecular system can be represented by a single global state (e.g., a Markov state in a Markov state model [MSM]). In this approach, molecules can be extensively sampled and analyzed when they only possess a few metastable states, such as small- to medium-sized proteins. However, this approach breaks down in frustrated systems and in large protein assemblies, where the number of global metastable states may grow exponentially with the system size. To address this problem, we here introduce dynamic graphical models (DGMs) that describe molecules as assemblies of coupled subsystems, akin to how spins interact in the Ising model. The change of each subsystem state is only governed by the states of itself and its neighbors. DGMs require fewer parameters than MSMs or other global state models; in particular, we do not need to observe all global system configurations to characterize them. Therefore, DGMs can predict previously unobserved molecular configurations. As a proof of concept, we demonstrate that DGMs can faithfully describe molecular thermodynamics and kinetics and predict previously unobserved metastable states for Ising models and protein simulations.

Understanding the methyl-TROSY effect over a wide range of magnetic fields.

The use of relaxation interference in the methyl Transverse Relaxation-Optimized SpectroscopY (TROSY) experiment has opened new avenues for the study of large proteins and protein assemblies in nuclear magnetic resonance. So far, the theoretical description of the methyl-TROSY experiment has been limited to the slow-tumbling approximation, which is correct for large proteins on high-field spectrometers. In a recent paper, favorable relaxation interference was observed in the methyl groups of a small protein at a magnetic field as low as 0.33 T, well outside the slow-tumbling regime. Here, we present a model to describe relaxation interference in methyl groups over a broad range of magnetic fields, not limited to the slow-tumbling regime. We predict that the type of multiple-quantum transition that shows favorable relaxation properties change with the magnetic field. Under the condition of fast methyl-group rotation, methyl-TROSY experiments can be recorded over the entire range of magnetic fields from a fraction of 1 T up to 100 T.