Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip.

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.

Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galß1-3GalNAc reactivity on Fetuin.

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galß1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galß1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004-2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galß1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galß1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galß1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galß1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.

A Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

A Soluble, Folded Protein without Charged Amino Acid Residues.

Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced.

Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus.

Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the -1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in K(M) for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, K(A), for the divalent metal ion (Co²âº, Zn²âº, Mn²âº, or Cd²âº). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn²âº concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH <4.5; therefore, only the basic limb of the bell-shaped pH profile was analyzed.

Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes.

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

Quantification of thiols and disulfides.

BACKGROUND: Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions. SCOPE OF REVIEW: In the present account, we briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the key chemical aspects of current methodology, together with identifying potential difficulties inherent in their experimental implementation. MAJOR CONCLUSIONS: While many reagents have been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox conditions in living cells presents a continuing challenge. GENERAL SIGNIFICANCE: Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while pool sizes of redox pairs and their intracellular distribution are being uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important aspect of cellular metabolism. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

The pKa value and accessibility of cysteine residues are key determinants for protein substrate discrimination by glutaredoxin.

The enzyme glutaredoxin catalyzes glutathione exchange, but little is known about its interaction with protein substrates. Very different proteins are substrates in vitro, and the enzyme seems to have low requirements for specific protein interactions. Here we present a systematic investigation of the interaction between human glutaredoxin 1 and glutathionylated variants of a single model protein. Thus, single cysteine variants of acyl-coenzyme A binding protein were produced creating a set of substrates in the same protein background. The rate constants for deglutathionylation differ by more than 2 orders of magnitude between the best (k1 = 1.75 × 10(5) M(-1) s(-1)) and the worst substrate (k1 = 4 × 10(2) M(-1) s(-1)). The pKa values of the substrate cysteine residues were determined by NMR spectroscopy and found to vary from 8.2 to 9.9. Rates of glutaredoxin 1-catalyzed deglutathionylation were assessed with respect to substrate cysteine pKa values, cysteine residue accessibility, local stability, and backbone dynamics. Good substrates are characterized by a combination of high accessibility of the glutathionylated site and low pKa of the cysteine residue.

Rapid TaqMan-based quantification of chlorophyll d-containing cyanobacteria in the genus Acaryochloris.

Reports of the chlorophyll (Chl) d-containing cyanobacterium Acaryochloris have accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution of Acaryochloris species was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection of Acaryochloris species in complex environmental samples. The TaqMan probe showed detection limits of ~10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from five Acaryochloris strains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of >94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence of Acaryochloris species in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chl d was confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates of Acaryochloris species amounted to 7.6 × 10(1) to 3.0 × 10(3) per mg of CCA. These numbers indicate a substantial contribution of Chl d-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence of Acaryochloris species and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.

Catalytic site interactions in yeast OMP synthase.

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.

Global Analysis of Multi-Mutants to Improve Protein Function.

The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to a previously published experiment reporting on >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions (Sarkisyan et al., 2016). The GMMA method achieves a good fit to this dataset while being analytically transparent. We show experimentally that the six top-ranking substitutions progressively enhance GFP. More broadly, using only a single experiment as input our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function. In conclusion, we suggest that large libraries of multiply-substituted variants may provide a unique source of information for protein engineering.

A fluorescent probe which allows highly specific thiol labeling at low pH.

Determination of the thiol-disulfide status in biological systems is challenging as redox pools are easily perturbed during sample preparation. This is particularly pertinent under neutral to mildly alkaline conditions typically required for alkylation of thiols. Here we describe the synthesis and properties of a thiol-specific reagent, fluorescent cyclic activated disulfide (FCAD), which includes the fluorescein moiety as fluorophore and utilizes a variation of thiol-disulfide exchange chemistry. The leaving-group character of FCAD makes it reactive at pH 3, allowing modification at low pH, limiting thiol-disulfide exchange. Different applications are demonstrated including picomolar thiol detection, determination of redox potentials, and in-gel detection of labeled proteins.

Quantifying the global cellular thiol-disulfide status.

It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data were combined with quantification of reduced and oxidized glutathione in the same cells. Of the total protein cysteines, 6% and 9.6% are engaged in disulfide bond formation in HEK and HeLa cells, respectively. Furthermore, the steady-state level of PSSG is <0.1% of the total protein cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress.

Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states.

Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover with a combination of NMR magnetization transfer, R(2) relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically tuned to exchange on the millisecond time scale. The conformational changes of the protein backbone during catalysis were followed by (15)N nuclear spin relaxation dispersion experiments. A conformational transition that is well described by a two-state process with an exchange rate corresponding to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions on the time scale of catalytic turnover were not observed for the enzyme in the resting states, implying that alternative conformers do not accumulate to significant concentrations. These results infer that the turnover rate in glutaredoxin is governed by formation of a productive enzyme-substrate encounter complex, and that catalysis proceeds by an induced fit mechanism rather than by conformer selection driven by intrinsic conformational dynamics.

Rational Protein Engineering to Increase the Activity and Stability of IsPETase Using the PROSS Algorithm.

Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.

Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles.

Luciferases are widely used as reporters for gene expression and for sensitive detection systems. The luciferase (GLuc) from the marine copepod Gaussia princeps, has gained popularity, primarily because it is secreted and displays a very high light intensity. While firefly luciferase is characterized by kinetic behavior which is consistent with conventional steady-state Michaelis-Menten kinetics, GLuc displays what has been termed "flash" kinetics, which signify a burst in light emission followed by a rapid decay. As the mechanistic background for this behavior was unclear, we decided to decipher this in more detail. We show that decay in light signal is not due to depletion of substrate, but rather is caused by the irreversible inactivation of the enzyme. Inactivation takes place after between 10 and 200 reaction cycles, depending on substrate concentration and can be described by the sum of two exponentials with associated rate constants. The dominant of these increases linearly with substrate concentration while the minor is substrate-concentration independent. In terms of rate of initial luminescence reaction, this increases with the substrate concentration to the power of 1.5 and shows no signs of saturation up to 10 µM coelenterazine. Finally, we find that the inactivated form of the enzyme has a larger apparent size in both size exclusion chromatography and SDS-PAGE analysis and shows a fluorescence peak at 410 nm when excited at 333 nm. These findings indicate that the "flash" kinetics in Gaussia luciferase are caused by an irreversible covalent binding to a substrate derivative during catalysis.

Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays.

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.