A new azobenzene-based design strategy for detergents in membrane protein research

Mass spectrometry enables the in-depth structural elucidation of membrane protein complexes, which is of great interest in structural biology and drug discovery. Recent breakthroughs in this field revealed the need for design rules that allow fine-tuning the properties of detergents in solution and gas phase. Desirable features include protein charge reduction, because it helps to preserve native features of protein complexes during transfer from solution into the vacuum of a mass spectrometer. Addressing this challenge, we here present the first systematic gas-phase study of azobenzene detergents. The utility of gas-phase techniques for monitoring light-driven changes of isomer ratios and molecular properties are investigated in detail. This leads to the first azobenzene detergent that enables the native mass spectrometry analysis of membrane proteins and whose charge-reducing properties can be tuned by irradiation with light. More broadly, the presented work outlines new avenues for the high-throughput characterization of supramolecular systems and opens a new design strategy for detergents in membrane protein research.

Here, L. H. Urner and co-workers identify a new detergent design strategy for the non-denaturing structural analysis of membrane proteins by studying the gas-phase properties of azobenzene-based oligoglycerol detergents.     

Single-molecule nanopore sensing of actin dynamics and drug binding

Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed the unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopore sensing for in-depth understanding of protein folding landscapes and for drug discovery.

Nanopipettes were used for real-time investigation into actin dynamics and drug binding at single-molecule resolution, showing promise for a better understanding of the mechanism of protein–protein interactions and drug discovery.     

Systematic quantification of the dynamics of newly synthesized proteins unveiling their degradation pathways in human cells

Proteins are continuously synthesized during cell growth and proliferation. At the same time, excessive and misfolded proteins have to be degraded, otherwise they are a burden to cells. Protein degradation is essential to maintain proteostasis in cells, and dysfunction of protein degradation systems results in numerous diseases such as cancer and neurodegenerative diseases. Despite the importance of protein degradation, the degradation pathways of many proteins remain to be explored. Here, we comprehensively investigated the degradation of newly synthesized proteins in human cells by integrating metabolic labeling, click chemistry, and multiplexed proteomics, and systematic and quantitative analysis of newly synthesized proteins first revealed the degradation pathways of many proteins. Bioinformatic analysis demonstrates that proteins degraded through two major pathways have distinct properties and functions. Proteins degraded through the ubiquitin-proteasome pathway contain more disordered structures, whereas those through the autophagy-lysosome pathway have significantly higher hydrophobicity. Systematic and quantitative investigation of the dynamics of newly synthesized proteins provides unprecedented and valuable information about protein degradation, which leads to a better understanding of protein properties and cellular activities.

Systematic quantification of the dynamics of newly synthesized proteins first reveals the degradation pathways of many proteins in human cells, and proteins degraded through each of the two major pathways have distinct properties and functions.     

Snapshotting the transient conformations and tracing the multiple pathways of single peptide folding using a solid-state nanopore

A fundamental question relating to protein folding/unfolding is the time evolution of the folding of a protein into its precisely defined native structure. The proper identification of transition conformations is essential for accurately describing the dynamic protein folding/unfolding pathways. Owing to the rapid transitions and sub-nm conformation differences involved, the acquisition of the transient conformations and dynamics of proteins is difficult due to limited instrumental resolution. Using the electrochemical confinement effect of a solid-state nanopore, we were able to snapshot the transient conformations and trace the multiple transition pathways of a single peptide inside a nanopore. By combining the results with a Markov chain model, this new single-molecule technique is applied to clarify the transition pathways of the β-hairpin peptide, which shows nonequilibrium fluctuations among several blockage current stages. This method enables the high-throughput investigation of transition pathways experimentally to access previously obscure peptide dynamics, which is significant for understanding the folding/unfolding mechanisms and misfolding of peptides or proteins.

A solid-state nanopore based method is described for resolving protein-folding-related problems via snapshotting the folding intermediates and characterizing the kinetics of a single peptide.     

NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

We solved the structure of a fluorogen-activating protein FAST and synthesized the library of potential fluorogens. Using these data, we designed the shortest genetically encoded fluorescent tag among all known.     

Unraveling the surface glycoprotein interaction network by integrating chemical crosslinking with MS-based proteomics

The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity. In this work, we developed a method integrating chemical crosslinking, an enzymatic reaction, and MS-based proteomics to systematically characterize proteins interacting with surface glycoproteins, and then constructed the surfaceome interaction network. Glycans covalently bound to proteins were employed as “baits”, and proteins that interact with surface glycoproteins were connected using chemical crosslinking. Glycans on surface glycoproteins were oxidized with galactose oxidase (GAO) and sequentially surface glycoproteins together with their interactors (“prey”) were enriched through hydrazide chemistry. In combination with quantitative proteomics, over 300 proteins interacting with surface glycoproteins were identified. Many important domains related to extracellular events were found on these proteins. Based on the protein–protein interaction database, we constructed the interaction network among the identified proteins, in which the hub proteins play more important roles in the interactome. Through analysis of crosslinked peptides, specific interactors were identified for glycoproteins on the cell surface. The newly developed method can be extensively applied to study glycoprotein interactions on the cell surface, including the dynamics of the surfaceome interactions in cells with external stimuli.

Proteins interacting with glycoproteins on the cell surface were systematically characterized by integrating chemical crosslinking, enzymatic oxidation, and MS-based proteomics. The surface glycoprotein interaction network was then constructed.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

A complete picture of protein unfolding and refolding in surfactants

Interactions between proteins and surfactants are of relevance in many applications including food, washing powder formulations, and drug formulation. The anionic surfactant sodium dodecyl sulfate (SDS) is known to unfold globular proteins, while the non-ionic surfactant octaethyleneglycol monododecyl ether (C₁₂E₈) can be used to refold proteins from their SDS-denatured state. While unfolding have been studied in detail at the protein level, a complete picture of the interplay between protein and surfactant in these processes is lacking. This gap in our knowledge is addressed in the current work, using the β-sheet-rich globular protein β-lactoglobulin (bLG). We combined stopped-flow time-resolved SAXS, fluorescence, and circular dichroism, respectively, to provide an unprecedented in-depth picture of the different steps involved in both protein unfolding and refolding in the presence of SDS and C₁₂E₈. During unfolding, core–shell bLG-SDS complexes were formed within ∼10 ms. This involved an initial rapid process where protein and SDS formed aggregates, followed by two slower processes, where the complexes first disaggregated into single protein structures situated asymmetrically on the SDS micelles, followed by isotropic redistribution of the protein. Refolding kinetics (>100 s) were slower than unfolding (<30 s), and involved rearrangements within the mixing deadtime (∼5 ms) and transient accumulation of unfolded monomeric protein, differing in structure from the original bLG-SDS structure. Refolding of bLG involved two steps: extraction of most of the SDS from the complexes followed by protein refolding. These results reveal that surfactant-mediated unfolding and refolding of proteins are complex processes with rearrangements occurring on time scales from sub-milliseconds to minutes.

The time-resolved study reveals several transition states during SDS-induced unfolding of the protein, as well as under refolding of the protein by the nonionic surfactant C₁₂E₈.     

Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.

The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass spectrometry instrumentation provides dramatically improved single-cell proteome profiling.     

Fast protein analysis enabled by high-temperature hydrolysis

While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the post-digestion treatment prior to mass spectrometry analysis. In this work, we developed an enzyme-free microreaction system for fast and selective hydrolysis of proteins, and a direct analysis of the protein digests was achieved by nanoESI (electrospray ionization) mass spectrometry. Using the microreactor, proteins in aqueous solution could be selectively hydrolyzed at the aspartyl sites within 2 min at high temperatures (∼150 °C). Being free of salts, the protein digest solution could be directly analyzed using a mass spectrometer with nanoESI without further purification or post-digestion treatment. This method has been validated for the analysis of a variety of proteins with molecular weights ranging from 8.5 to 67 kDa. With introduction of a reducing agent into the protein solutions, fast cleavage of disulfide bonds was also achieved along with high-temperature hydrolysis, allowing for fast analysis of large proteins such as bovine serum albumin. The high-temperature microreaction system was also used with a miniature mass spectrometer for the determination of highly specific peptides from Mycobacterium tuberculosis antigens, showing its potential for point-of-care analysis of protein biomarkers.

A high-temperature microreaction system is developed for fast and selective hydrolysis of proteins, enabling direct analysis of protein biomarkers by mass spectrometry.     

A straightforward methodology to overcome solubility challenges for N-terminal cysteinyl peptide segments used in native chemical ligation

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2.

We herein describe a straightforward approach for the introduction of a solubilizing tag on N-terminal cysteinyl segments used in native chemical ligation-based protein chemical synthesis. Conveniently, the tag is removed during the ligation.     

Identifying reactive intermediates by mass spectrometry

Development of new reactions requires finding and understanding of novel reaction pathways. In challenging reactions such as C–H activations, these pathways often involve highly reactive intermediates which are the key to our understanding, but difficult to study. Mass spectrometry has a unique sensitivity for detecting low abundant charged species; therefore it is increasingly used for detection of such intermediates in metal catalysed- and organometallic reactions. This perspective shows recent developments in the field of mass spectrometric research of reaction mechanisms with a special focus on going beyond mass-detection. Chapters discuss the advantages of collision-induced dissociation, ion mobility and ion spectroscopy for characterization of structures of the detected intermediates. In addition, we discuss the relationship between the condensed phase chemistry and mass spectrometric detection of species from solution.

Modern approaches of mass spectrometry can identify reaction intermediates and provide a unique insight into their structure, properties and kinetics.     

Interplay between intrinsically disordered proteins inside membraneless protein liquid droplets

Intrinsically disordered proteins (IDPs) in cells phase separate to form diverse membraneless organelles, which have condensed liquid droplet-like properties and often contain multiple IDPs. However, how potential interactions between different IDPs affect the dynamic behavior of these protein droplets is largely unknown. Here, we develop a rapid IDP clustering system to generate protein droplets with varied residue compositions and examine diverse interacting IDPs inside droplets. Three different IDP droplets actively recruited other diverse IDPs inside droplets with extremely varied enrichment (inside/outside) degrees (over 100-fold variation) under highly crowded conditions. The recruited IDPs were mostly mobile even inside highly immobile droplets. Among the five tested IDPs, the disordered region of Ddx4 helicase with its unique multiple charged residue blocks was noticeably influenced by droplet mobility. We also discovered that droplets of different IDPs could rapidly fuse to each other. Interestingly, some droplets were heterogeneously fused with segregated subcompartments, and this segregation was enhanced by droplet maturation and was more apparent for specific IDP pairs, in which the polar and charged residue compositions are highly different. The present study not only reports multiple peculiar behaviors of interacting IDP pairs inside droplets but also provides valuable information on generating membraneless organelle models with controllable droplet properties.

Membraneless droplets of intrinsically disordered proteins (IDPs) with varied residue compositions uniquely interact with each other as droplets and clients.     

Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins

The conservation of chemoselectivity becomes invalid for multiple electrophilic warheads during protein bioconjugation. Consequently, it leads to unpredictable heterogeneous labeling of proteins. Here, we report that a linchpin can create a unique chemical space to enable site-selectivity for histidine and aspartic acid modifications overcoming the pre-requisite of chemoselectivity.

Linchpin-enabled promiscuous electrophile uncovers an unchartered reactivity landscape for the precision engineering of proteins.     

Proteomimetic surface fragments distinguish targets by function

The fragment-centric design promises a means to develop complex xenobiotic protein surface mimetics, but it is challenging to find locally biomimetic structures. To address this issue, foldameric local surface mimetic (LSM) libraries were constructed. Protein affinity patterns, ligand promiscuity and protein druggability were evaluated using pull-down data for targets with various interaction tendencies and levels of homology. LSM probes based on H14 helices exhibited sufficient binding affinities for the detection of both orthosteric and non-orthosteric spots, and overall binding tendencies correlated with the magnitude of the target interactome. Binding was driven by two proteinogenic side chains and LSM probes could distinguish structurally similar proteins with different functions, indicating limited promiscuity. Binding patterns displayed similar side chain enrichment values to those for native protein–protein interfaces implying locally biomimetic behavior. These analyses suggest that in a fragment-centric approach foldameric LSMs can serve as useful probes and building blocks for undruggable protein interfaces.

Foldameric local surface mimetics (LSMs) detect spots at protein surfaces and are promising building blocks in a fragment-centric design of xenobiotic structures and protein–protein interaction inhibitors.     

Exploring conformational preferences of proteins: ionic liquid effects on the energy landscape of avidin

In this work we experimentally investigate solvent and temperature induced conformational transitions of proteins and examine the role of ion–protein interactions in determining the conformational preferences of avidin, a homotetrameric glycoprotein, in choline-based ionic liquid (IL) solutions. Avidin was modified by surface cationisation and the addition of anionic surfactants, and the structural, thermal, and conformational stabilities of native and modified avidin were examined using dynamic light scattering, differential scanning calorimetry, and thermogravimetric analysis experiments. The protein-surfactant nanoconjugates showed higher thermostability behaviour compared to unmodified avidin, demonstrating distinct conformational ensembles. Small-angle X-ray scattering data showed that with increasing IL concentration, avidin became more compact, interpreted in the context of molecular confinement. To experimentally determine the detailed effects of IL on the energy landscape of avidin, differential scanning fluorimetry and variable temperature circular dichroism spectroscopy were performed. We show that different IL solutions can influence avidin conformation and thermal stability, and we provide insight into the effects of ILs on the folding pathways and thermodynamics of proteins. To further study the effects of ILs on avidin binding and correlate thermostability with conformational heterogeneity, we conducted a binding study. We found the ILs examined inhibited ligand binding in native avidin while enhancing binding in the modified protein, indicating ILs can influence the conformational stability of the distinct proteins differently. Significantly, this work presents a systematic strategy to explore protein conformational space and experimentally detect and characterise ‘invisible’ rare conformations using ILs.

Revealing solvent and temperature induced conformational transitions of proteins and the role of ion–protein interactions in determining the conformational preferences of avidin in ionic liquids.     

Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein–ligand complex structure analysis

Room-temperature (RT) protein crystallography provides significant information to elucidate protein function under physiological conditions. In particular, contrary to typical binding assays, X-ray crystal structure analysis of a protein–ligand complex can determine the three-dimensional (3D) configuration of its binding site. This allows the development of effective drugs by structure-based and fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein–ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein–ligand complex analysis, the simultaneous preparation of protein–ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein–ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein–ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin–ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.

Room temperature protein crystallography and its application to protein–ligand complex structure analysis was demonstrated using a microfluidic protein crystal array device.     

A single amino acid Gly-tag enables metal-free protein purification

Analytically pure proteins are indispensable for diverse applications, including therapeutics. Here, we report a methodology where a single amino acid, glycine, enables metal-free protein purification. This robust platform is enabled by a Gly-tag resin for site-specific capture, enrichment, and release through chemically triggered C–C bond dissociation by resonance-assisted electron density polarization.

Gly-tag resin precisely captures and releases a protein with one glycine at the N-terminus. The user-friendly protocol delivers analytically pure protein free of metal contaminants.     

Fungal siderophore biosynthesis catalysed by an iterative nonribosomal peptide synthetase

Siderophores play a vital role in the viability of fungi and are essential for the virulence of many pathogenic fungal species. Despite their importance in fungal physiology and pathogenesis, the programming rule of siderophore assembly by fungal nonribosomal peptide synthetases (NRPSs) remains unresolved. Here, we report the characterization of the bimodular fungal NRPS, SidD, responsible for construction of the extracellular siderophore fusarinine C. The use of intact protein mass spectrometry, together with in vitro biochemical assays of native and dissected enzymes, provided snapshots of individual biosynthetic steps during NPRS catalysis. The adenylation and condensation domain of SidD can iteratively load and condense the amino acid building block cis-AMHO, respectively, to synthesize fusarinine C. Our study showcases the iterative programming features of fungal siderophore-producing NRPSs.

Snapshots of fungal siderophore biosynthesis on the biosynthetic assembly-line captured by intact protein mass-spectrometry.