Biology subjects

Winther, J. R.

Publications and source records attributed to Winther, J. R..

12 recordsLinked to original sources

bioRxiv2025

A highly stable engineered disulfide bond in the dimer interface of E. coli orotate phosphoribosyl transferase

Structural disulfides are very rarely found in cytoplasmic proteins of mesophilic organisms. Nevertheless, disulfides can form in the cytosol if these are stabilized sufficiently by the supporting protein structure. To investigate the redox properties of structural disulfide bonds, we introduced disulfide bonds into the cytosolic enzyme from E. coli, orotate phosphoribosyl transferase (OPRT). Because OPRT is a homodimer the introduction of opposing cysteine residues (R44C and D92C) into separate monomers of the enzyme meant that disulfide bond formation could easily be followed by non-reducing SDS-PAGE. Redox titration experiments in DTT buffers revealed that the designed disulfide bond was exceedingly stable with a redox potential of -314 mV, close to that of dithiothreitol (DTT). This interchain disulfide bond improved global thermostability by 5.9 {degrees}C relative to wild type protein and 21.4 {degrees}C above its reduced form. The catalytic activity of the enzyme in either its thiol or disulfide state was not substantially affected, indicating structural integrity. Combining an inactive subunit with the asymmetric nature of the disulfide bond enabled determining the rates of subunit rearrangement and disulfide formation. The R44C/D92C double mutant resulted in the formation of a symmetric homodimer with two interchain disulfides. However, the global stability of the two-disulfide dimer was not substantially increased relative to the dimer with the single disulfide bond, suggesting that the stabilizing effect was gained by preventing dimer dissociation. Finally, we show that the engineered disulfide bonds are formed to a significant degree in the cytosol of yeast cells, allowing us to determine the redox potential of the cytosol in equilibrium with this substrate to -300 mV. This is slightly lower than that previously determined using a GFP-based sensor, rxYFP in yeast. We find that disulfide bond formation is particularly enhanced in mutants lacking the glutathione reductase, Glr1. Thus, the engineered disulfide provides an alternative method for determining intracellular redox potential in living cells with an extended dynamic range relative to GFP-based sensors.

biochemistry↗

bioRxiv2024

Systematic characterization of indel variants using a yeast-based protein folding sensor

Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet compared to missense variants the effects of indels are poorly understood and predicted. To approach this issue, we developed a sensitive protein folding sensor based on complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor accurately reports on the folding of disease-linked missense variants and de novo designed proteins. Applying the folding sensor to a saturated library of single amino acid indel variants in human DHFR revealed that most regions which tolerate indels are confined to internal loops and the N- and C-termini. Surprisingly, indels are also allowed at a central -helix. Several indels are temperature-sensitive and the folding of most of these indels is rescued upon binding to the competitive DHFR inhibitor methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels operate by destabilizing the native fold and that these computational tools may be useful for classification of indels observed in population sequencing.

biochemistry↗

bioRxiv2023

A suicidal and extensively disordered luciferase with a bright luminescence

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 [A] and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase. Significance statementEnzymes are typically characterized by an overall globular structure with a hydrophobic core and a defined cavity for binding of substrate, containing the active site amino acid residues. Gaussia Luciferase is a widely used luminescent reporter with a very strong, albeit short-lived, flash of light due to rapid auto-inactivation. We show, using solution NMR, that while this luciferase shows some secondary structure elements held together by disulfide bonds this highly unusual enzyme is extensively disordered with essentially no hydrophobic core. Although the enzymatic mechanism remains unknown, we have identified two essential arginine residues but, in the structure, these do not point into a common active site. In spite of this, the enzyme has high substrate specificity suggesting that it undergoes major structural rearrangements upon binding of substrate.

biochemistry↗

bioRxiv2022

Increasing protein stability by inferring substitution effects from high-throughput experiments

Protein stability is an important parameter in almost all protein-engineering efforts. Evaluating the effects of the many possible amino acid changes to guide such projects is a significant task, even with recent advances in experimental and computational approaches. Here, we apply a computational model, GMMA, to extract substitution effects from a cost-effective genetic screen of a randomly mutated protein library. Using a high mutation frequency, the method can map stability effects of even very stable proteins for which conventional selection systems have reached their limit. Thus, we screened a mutant library of a highly stable and optimised model protein using an in vivo genetic sensor for folding and assigned a stability effect to 374 of 912 possible single amino acid substitutions. Combining the top 9 substitutions increased the thermodynamic stability by almost 50% in a single engineering step. This illustrates the capability of the method, which is applicable to any screen for protein function.

biochemistry↗

bioRxiv2021

Prolonging coagulant activity of factor Xa under hemophilic conditions by site-specific N-glycosylation of the surface-exposed autolysis loop

The regulation of Factor X (FX) is critical to maintain hemostasis. To gain insights to the regulation of the active and zymogen form of coagulation FX, we probed specific molecular interactions by introducing novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. Introduction of N-glycans in the autolysis loop of these FX variants decreased Factor VIIa (FVIIa)-mediated activation ~3-fold and prothrombin activation 2- to 10-fold presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor Factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, while ATIII inhibition in the presence of the heparin-analogue Fondaparinux was modestly increased by 2- to 15-fold compared to wild type. To probe the in vitro hemostatic effect of the FX variants, the thrombin generation potential in FX-depleted plasma was evaluated. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FX variants with N-glycosylation at positions 148-150. These results indicate that residues of the surface-exposed autolysis loop and residues close by participate in FX activation, proteolytic activity and inhibition of FXa by TFPI and ATIII. In plasma-based assays, a modest decrease in FX-activation rate appeared to compensate for the collective reduction in inhibitor interactions.

biochemistry↗

bioRxiv2021

Computational and experimental assessment of backbone templates for computational protein design

Computational protein design has taken big strides over the recent years, however, the tools available are still not at a state where a sequence can be designed to fold into a given protein structure at will and with high probability. We have here applied a recent release of Rosetta Design to redesign a set of structurally very similar proteins belonging to the Thioredoxin fold. We determined design success using a combination of a genetic screening tool to assay folding/stability in E. coli and selecting the best hits from this for further biochemical characterization. We have previously used this set of template proteins for redesign and found that success was highly dependent on template structure, a trait which was also found in this study. Nevertheless, state of the art design software is now able to predict the best template, most likely due to the introduction of the cart_bonded energy term. The template that led to the greatest fraction of successful designs was the same (a Thioredoxin from spinach) as that identified in our previous study. Our previously described redesign of Thioredoxin, which also used the spinach protein as template, however also performed well. In the present study, both these templates yielded proteins with compact folded structures, and enforces the conclusion that any design project must carefully consider different design templates. Fortunately, selecting designs using the cart_bonded energy term appears to correctly identify such templates.

biochemistry↗

bioRxiv2020

Global analysis of multi-mutants to discover stabilizing amino acid substitutions

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 >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions. The GMMA method achieves a good fit to the data while being analytically transparent. The six top-ranking substitutions are demonstrated to progressively enhance GFP and in general, our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function, only using a single experiment as input. Significance StatementProtein engineering is carried out to improve proteins for practical applications by changing one or more amino acid residues in a protein. We present a method termed global multi-mutant analysis (GMMA) that helps solve two problems in protein engineering. First, because many proteins are already highly optimized, it can be difficult to identify individual variants that further improve function. Second, while it is possible to combine variants with small individual effects, such approaches may be hampered by non-additivity. GMMA identifies combinable effects of single substitutions from a large set of variants each carrying multiple substitutions. We demonstrate the approach on a set of 54,000 variants of green fluorescent protein and identify many enhancing single-substitutions from a single experiment.

biophysics↗

bioRxiv2020

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. While luciferases from firefly and Renilla have long been used for analysis of intracellular expression, the luciferase (GLuc) from the marine copepod Gaussia princeps, has gained popularity, primarily because it is secreted and displays a very high light intensity. Firefly luciferase is characterized by kinetic behavior which is consistent with conventional steady-state Michaelis-Menten kinetics (termed "glow" kinetics). GLuc, conversely, 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 is poorly characterized, we decided to decipher the mechanism 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. We found that the rate of inactivation is described by the sum of two exponentials with associated rate constants. The dominant of these 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 1.53 and shows no signs of saturation up to 10 M coelenterazine. Finally, we found 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 derivative of the substrate during the reaction.

molecular biology↗

bioRxiv2020

Charge Interactions in a Highly Charge-depleted Protein

Interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations, and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions. SignificanceElectrostatic forces are important for protein folding and are favored targets of protein engineering. However, despite the many advances in the field of protein electrostatics, the prediction of changes in protein structure and function upon introduction or removal of titratable residues is still complicated. In order to provide a basic understanding of protein electrostatics we here characterize a highly charge-depleted protein and its titratable variants by a combination of NMR spectroscopy and constant pH molecular dynamics simulations. Our investigations reveal how strongly interacting residues engaged in salt bridging, can be characterized. Furthermore, our study may also enrich and facilitate the understanding of dehydration of salt-bridges and its potential effect on protein stability.

biochemistry↗

bioRxiv2020

Substitutional landscape of a split fluorescent protein fragment

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 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 strand 10 variants 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 and 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 determination of robustness and precision of the method. We showed that the microarray fluorescence correlated with the affinity in solution between the LOO-FP and peptides. A double substitution yielded a peptide with 9-fold higher affinity than the starting peptide.

synthetic biology↗

bioRxiv2019

Mutations in a single signaling pathway allow growth on a different solvent than water

Since life is completely dependent on water, it is difficult to gauge the impact of solvent change. To analyze the role of water as a solvent in biology, we replaced water with heavy water (D2O), and investigated the biological effects by a wide range of techniques, using the fission yeast Schizosaccharomyces pombe as model organism. We show that high concentrations of D2O lead to altered glucose metabolism, growth retardation, and inhibition of meiosis. However, mitosis and overall cell viability were only slightly affected. After prolonged incubation in D2O, cells displayed gross morphological changes, thickened cell walls as well as aberrant septa and cytoskeletal organization. RNA sequencing revealed that D2O causes a strong downregulation of most tRNAs and triggers activation of the general stress response pathway. Genetic screens identified several D2O sensitive mutants, while mutants compromised in the cell integrity pathway, including the protein kinase genes pmk1, mkh1, pek1 and pck2, that control cell wall biogenesis, were more tolerant to D2O. We speculate that D2O affects the phospholipid membrane or cell wall glycans causing an activation of the cell integrity pathway. In conclusion, the effects of solvent replacement are pleiotropic but the D2O-triggered activation of the cell integrity pathway and subsequent increased deposition of cell wall material and septation problems appear most critical for the cell growth defects.

genetics↗

bioRxiv2018

Deep mutational scanning by FACS-sorting of encapsulated E. coli micro-colonies

We present a method for high-throughput screening of protein variants where the signal is enhanced by micro-encapsulation of single cells into 20-30 m agarose beads. Cells inside beads are propagated using standard agitation in liquid media and grow clonally into micro-colonies harboring several hundred bacteria. We have, as a proof-of-concept, analyzed random amino acid substitutions in the five C-terminal {beta}-strands of the Green Fluorescent Protein (GFP). Starting from libraries of variants, each bead represents a clonal line of cells that can be separated by Fluorescence Activated Cell Sorting (FACS). Pools representing collections of individual variants with desired properties are subsequently analyzed by deep sequencing. Notably, the encapsulation approach described holds the potential for high-throughput analysis of systems where the fluorescence signal from a single cell is insufficient for detection. Fusion to GFP, or use of fluorogenic substrates, allows coupling protein levels or activity to sequence for a wide range of proteins. Here we analyzed more than 10,000 individual variants to gauge the effect of mutations on GFP-fluorescence. In the mutated region, we observed virtually all amino acid substitutions that are accessible by single nucleotide exchange. Lastly, we assessed the performance of biophysical protein stability predictors, FoldX and Rosetta, in predicting the outcome of the experiment. Both tools display good performance on average, suggesting that loss of thermodynamic stability is a key mechanism for the observed variation of the mutants. This, in turn, suggests that deep mutational scanning datasets may be used to more efficiently fine-tune such predictors, especially for mutations poorly covered by current biophysical data.

bioengineering↗