CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

Selenoprotein K (selenok) is linked to the integrated stress response, which helps cells combat stressors and regain normal function. The selenoprotein contains numerous protein interaction hubs and post-translational modification sites and is involved in protein palmitoylation, vesicle trafficking, and the resolution of ER stress. Anchored to the endoplasmic reticulum (ER) membrane, selenok interacts with protein partners to influence their stability, localization, and trafficking, impacting various cellular functions such as calcium homeostasis, cellular migration, phagocytosis, gene expression, and immune response. Consequently, selenok expression level is linked to cancer and neurodegenerative diseases.

Because it contains the reactive amino acid selenocysteine, selenok is likely to function as an enzyme. However, highly unusual for enzymes, the protein segment containing the selenocysteine lacks a stable secondary or tertiary structure, yet it includes multiple interaction sites for protein partners and post-translational modifications. Currently, the reason(s) for the presence of the rare selenocysteine in selenok is not known. Furthermore, of selenok’s numerous interaction sites, only some have been sufficiently characterized, leaving many of selenok’s potential protein partners to be discovered. In this review, we explore selenok’s role in various cellular pathways and its impact on human health, thereby highlighting the links between its diverse cellular functions.

NMR spectroscopy has been applied to virtually all sites within proteins and biomolecules; however, the observation of sulfur sites remains very challenging. Recent studies have examined ⁷⁷Se as a replacement for sulfur and applied ⁷⁷Se NMR in both the solution and solid states. As a spin-1/2 nuclide, ⁷⁷Se is attractive as a probe of sulfur sites, and it has a very large chemical shift range (due to a large chemical shift anisotropy), which makes it potentially very sensitive to structural and/or binding interactions as well as dynamics. Despite being a spin-1/2 nuclide, there have been rather limited studies of ⁷⁷Se, and the ability to use ¹H-indirect detection has been sparse. Some examples exist, but in the absence of a directly bonded, nonexchangeable ¹H, these have been largely limited to smaller molecules. We develop and illustrate approaches using double-labeling of ¹³C and ⁷⁷Se in proteins that enable more sensitive triple-resonance schemes via multistep coherence transfers and ¹H-detection. These methods require specialized hardware and decoupling schemes, which we developed and will be discussed.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replicates and evades detection using ER membranes and their associated protein machinery. Among these hijacked human proteins is selenoprotein S. This selenoprotein takes part in the protein quality control, NF-kB signaling, and the regulation of cytokine secretion. While the role of selenos in the viral life cycle is not yet known, it has been reported to interact with SARS-CoV-2 nonstructural protein 7 (nsp7), a viral protein essential for the replication of the virus. We set to study whether selenos and nsp7 interact directly and if they can still bind when nsp7 is bound to the replication and transcription complex of the virus. Using biochemical assays, we show that selenos binds directly to nsp7. In addition, we found that selenos can bind to nsp7 when it is in a complex with the coronavirus’s minimal replication complex, comprised of nsp7, nsp8, and the RNA-dependent RNA polymerase nsp12. In addition, through crosslinking experiments, we mapped the interaction sites of selenoprotein S and nsp7 in the replication complex and showed that the hydrophobic segment of selenos is essential for binding to nsp7. This arrangement leaves an extended helix and the intrinsically disordered segment of selenos¾including the reactive selenocysteine¾exposed and free to potentially recruit additional proteins to the replication and transcription complex. Thus, selenoprotein S directly interacts with the three viral proteins that constitute the core of the virus’s replication and transcription complex.

Selenoprotein S (selenos) is a small, intrinsically disordered membrane protein that is associated with various cellular functions, such as inflammatory processes, cellular stress response, protein quality control, and signaling pathways. It is primarily known for its contribution to the ER-associated degradation (ERAD) pathway, which governs the extraction of misfolded proteins or misassembled protein complexes from the ER to the cytosol for degradation by the proteasome. However, selenos’s other cellular roles in signaling are equally vital, including the control of transcription factors and cytokine levels. Consequently, genetic polymorphisms of selenos are associated with increased risk for diabetes, dyslipidemia, and cardiovascular diseases, while high expression levels correlate with poor prognosis in several cancers. Its inhibitory role in cytokine secretion is also exploited by viruses. Since selenos binds multiple protein complexes, however, its specific contributions to various cellular pathways and diseases have been difficult to establish. Thus, the precise cellular functions of selenos and their interconnectivity have only recently begun to emerge. This review aims to summarize recent insights into the structure, interactome, and cellular roles of selenos.

The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen Candida albicans also uses an LRR domain-containing protein, Cyrp1, to sense bacterial cell wall fragments. However, the dynamics of signaling and detection of bacterial peptidoglycan fragments by the LRR of Cyr1p remains poorly characterized. Here we develop optimal recombinant expression workflows and provide characterization of the entire region of the LRR domain of Cyr1p as a peripheral membrane protein. Using a newly designed peptidoglycan enrichment bead assay, we demonstrate that this domain can bind bacterial peptidoglycan fragments under native conditions. The new membrane-associated Cyr1p−LRR construct sets the stage for the development of antifungal agents via high-throughput campaigns to inhibit cell wall−Cyr1p interactions.

The Franklin Institute awards the Benjamin Franklin Medal in Chemistry to Professor Dame Carol V. Robinson for developing techniques to analyze the interactions of biological molecules such as proteins and lipids and thereby elucidate their biological functions and assist in pharmaceutical discovery.

Professor Robinson is recognized as a key figure and driving force in the development of native mass spectrometry, which has become an essential tool in the study of proteins’ structure, function, and interactions with other molecules. Due to her keen insights, diligent work, and unwavering scientific approach, she has turned ideas that once were met with strong resistance, into today’s well-known facts. The first such discovery is that, under the proper conditions, biological macromolecules can be stripped of their surrounding water and lipid molecules without losing their native, and thus biological relevant, states. This is the core principle that underpins native mass spectrometry and its importance for the life sciences and human health-related research. Furthermore, Professor Robinson has led the key efforts that unequivocally demonstrated that this core principle also extends to large, multi-protein complexes. Toward that end, her team significantly expanded the overall capabilities of biological mass spectrometry by developing new methodologies, hardware, and software. Through this work, she paved the way for a new and powerful approach that enables the detailed study of large assemblies of macromolecules. Professor Robinson’s research must also be credited for groundbreaking work on membrane proteins. Using a fine-tuned mass spectrometry approach, she discovered and identified various roles of lipids in the coordination, binding, and activation of membrane proteins. Most recently, she demonstrated that it is possible to transfer proteins from their cellular environment into the mass spectrometer without intermediate steps that require specialized chemicals. Because these steps can introduce artifacts in measurements, analysis, and data interpretation, their absence resulted in data of unprecedented quality and information content.

Professor Robinson is widely known as an exceptional educator who has mentored countless scientists, many with exceptional careers in academia and industry. As a scientist with an unconventional career trajectory, she has been a tireless advocate for women in science and an inspiration for many young minds.

Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coliand mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme–substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

Objective: T cell activation triggers metabolic reprogramming to meet increased demand for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cell and cancer cells, which led us to investigate a potential role during T cell activation.

Methods: Since selenoprotein I (SELENOI) is an enzyme participating in two synthesis pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. Results: SELENOI knockout (KO) in mouse T cells exhibited reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor, AMPK. AMPK is inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment. Conclusions: T cell activation upregulates SELENOI dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.

Nature expands its chemical repertoire by employing the chalcogens sulfur and selenium. The two share many properties but because selenium has more electrons, those in its valence shell are more loosely held than sulfur’s and hence more reactive. As a result, selenium is not only a better nucleophile but also forms stronger intramolecular bonds (Reich and Hondal, 2016). To take advantage of these properties and enable enzymatic mechanisms with enhanced reactivity and higher efficiency, nature has evolved to produce selenoproteins, i.e., proteins that contain selenium (Arnér, 2010). Unlike metals or organic co-factors, selenium incorporates into the cellular makeup in the form of the rare amino acid selenocysteine (Sec, U). Sec is inserted at the designated position during selenoprotein synthesis by the ribosome. Selenoproteins carry out important functions in a variety of diverse pathways, such as signaling and detoxification of reactive oxygen species, regulation of redox pathways, the synthesis of hormones, calcium signaling and the unfolded protein response (Hatfield et al., 2014). Besides its direct biological relevance Sec can also serve as a selective tool in protein engineering as it is a convenient way to introduce site-specific modifications, direct folding or form diselenide bridges that are stable even under harsh conditions. Because of this ever growing scientific and technological interest in native and engineered selenoproteins, the reliable, high-yield production of these proteins has been much desired. However, the high reactivity of selenium and its compounds comes at the price of their substantial cell toxicity if left unchecked. Thus, it comes as no surprise that the synthesis and insertion of Sec are tightly controlled by the cell and optimized for low overall selenium flux. This is achieved by a variety of measures detailed later, but together they render Sec’s path into a protein significantly different from that of any canonical amino acid. It is these special features of the tightly controlled Sec insertion together with selenium’s toxicity that have posed as the major challenges to the large-scale production of native and engineered selenoproteins. Yet, employing innovative methods, from rational design of the insertion machinery to the optimization of whole genomes, these challenges are creatively addressed. Here, we provide an overview of these chemical biology efforts to optimize site-specific Sec incorporation for selenoprotein production.

Significance

In redox biology, the chemistry performed by proteins that contain the rare amino acid selenocysteine is frequently critical to the detoxification of reactive species that are harmful to cellular function. Selenocysteine and cysteine partner to form a motif featuring a sulfur–selenium covalent bond in many selenoproteins. This work demonstrates that selenium NMR, when paired with calculations, can provide critical insight concerning the local environment of these enigmatic redox motifs. It details how redox potentials, conformational preferences, and mobilities of such redox motifs change when the local environment of the selenocysteine is varied. Surprisingly, reverting selenocysteine to cysteine exerts only minor effects on redox potential. These new approaches deepen our understanding of the chemical reactivity and thermodynamic properties of selenoenzymes.

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable ⁷⁷Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate ⁷⁷Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidaseaugmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, ⁷⁷Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.