Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites, and a few nonconventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is nonessential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1.

WSe2 Light-Emitting Device Coupled to an h-BN Waveguide.

Optical information processing using photonic integrated circuits is a key goal in the field of nanophotonics. Extensive research efforts have led to remarkable progress in integrating active and passive device functionalities within one single photonic circuit. Still, to date, one of the central components, i.e., light sources, remain a challenge to be integrated. Here, we focus on a photonic platform that is solely based on two-dimensional materials to enable the integration of electrically contacted optoelectronic devices inside the light-confining dielectric of photonic structures. We combine light-emitting devices, based on exciton recombination in transition metal dichalcogenides, with hexagonal boron nitride photonic waveguides in a single van der Waals heterostructure. Waveguide-coupled light emission is achieved by sandwiching the light-emitting device between two hexagonal boron nitride slabs and patterning the complete van der Waals stack into a photonic structure. Our demonstration of on-chip light generation and waveguiding is a key component for future integrated van der Waals optoelectronics.

Polarization and Phase Textures in Lattice Plasmon Condensates.

Polarization textures of light may reflect fundamental phenomena, such as topological defects, and can be utilized in engineering light beams. They have been observed, for instance, in photonic crystal lasers and semiconductor polariton condensates. Here we demonstrate domain wall polarization textures in a plasmonic lattice Bose-Einstein condensate. A key ingredient of the textures is found to be a condensate phase that varies spatially in a nontrivial manner. The phase of the Bose-Einstein condensate is reconstructed from the real- and Fourier-space images using a phase retrieval algorithm. We introduce a simple theoretical model that captures the results and can be used for design of the polarization patterns and demonstrate that the textures can be optically switched. The results open new prospects for fundamental studies of non-equilibrium condensation and sources of polarization-structured beams.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.

Efficient soluble production of folded cat allergen Fel d 1 in Escherichia coli.

The major cat allergen Fel d 1 is one of the most common and potent causes of animal related allergy. Medical treatment of cat allergy has relied on immunotherapy carried out with cat dander extract. This approach has been problematic, mainly due to inconsistent levels of the major allergen in the produced extracts. Recombinant DNA technology has been proposed as an alternative method to produce more consistent pharmaceuticals for immunotherapy and diagnostics of allergy. Current approaches to produce recombinant Fel d 1 (recFel d 1) in the cytoplasm of Escherichia coli have however resulted in protein folding deficiencies and insoluble inclusion body formation, requiring elaborate in vitro processing to acquire folded material. In this study, we introduce an efficient method for cytoplasmic production of recFel d 1 that utilizes eukaryotic folding factors to aid recFel d 1 to fold and be produced in the soluble fraction of E. coli. The solubly expressed recFel d 1 is shown by biophysical in vitro experiments to contain structural disulfides, is extremely stable, and has a sensitivity for methionine sulfoxidation. The latter is discussed in the context of functional relevance.

Spatial and Temporal Coherence in Strongly Coupled Plasmonic Bose-Einstein Condensates.

We report first-order spatial and temporal correlations in strongly coupled plasmonic Bose-Einstein condensates. The condensate is large, more than 20 times the spatial coherence length of the polaritons in the uncondensed system and 100 times the healing length, making plasmonic lattices an attractive platform for studying long-range spatial correlations in two dimensions. We find that both spatial and temporal coherence display nonexponential decay; the results suggest power-law or stretched exponential behavior with different exponents for spatial and temporal correlation decays.

Multimode Organic Polariton Lasing.

We present a beyond-mean-field approach to predict the nature of organic polariton lasing, accounting for all relevant photon modes in a planar microcavity. Starting from a microscopic picture, we show how lasing can switch between polaritonic states resonant with the maximal gain, and those at the bottom of the polariton dispersion. We show how the population of nonlasing modes can be found, and by using two-time correlations, we show how the photoluminescence spectrum (of both lasing and nonlasing modes) evolves with pumping and coupling strength, confirming recent experimental work on the origin of blueshift for polariton lasing.

Sub-picosecond thermalization dynamics in condensation of strongly coupled lattice plasmons.

Bosonic condensates offer exciting prospects for studies of non-equilibrium quantum dynamics. Understanding the dynamics is particularly challenging in the sub-picosecond timescales typical for room temperature luminous driven-dissipative condensates. Here we combine a lattice of plasmonic nanoparticles with dye molecule solution at the strong coupling regime, and pump the molecules optically. The emitted light reveals three distinct regimes: one-dimensional lasing, incomplete stimulated thermalization, and two-dimensional multimode condensation. The condensate is achieved by matching the thermalization rate with the lattice size and occurs only for pump pulse durations below a critical value. Our results give access to control and monitoring of thermalization processes and condensate formation at sub-picosecond timescale.

Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)-protein disulfide-isomerase relay when protein folding capacity is exceeded.

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD). The UPR is triggered by unfolded protein stress and leads to up-regulation of cellular components such as chaperones and folding catalysts. These components relieve stress by increasing folding capacity and up-regulating ERAD components that remove non-native proteins. Although oxidative protein folding and the UPR/ERAD pathways each are well-understood, very little is known about any direct cross-talk between them. In this study, we carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfide-isomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation-reduction cycles and may also enhance ERAD, which requires reduced protein substrates. Relief from excess levels of non-native proteins by increasing the levels of folding factors removed the feedback inhibition. These results reveal regulatory cross-talk between the oxidative protein folding and UPR and ERAD pathways.

Efficient production of wild-type lipase B from Candida antarctica in the cytoplasm of Escherichia coli.

Candida antarctica lipase B (CalB) is a very efficient catalyst and is used in a wide range of industries from food flavour to pharmaceutical, and biodiesel manufacturing. It has a high degree of enantioselective and regioselective substrate specificity and is stable over a wide range of biophysical conditions including pH, temperature and solvent conditions. High-level expression of biologically active wild-type CalB has been problematic, partly due to folding events. Consequently, focus has been on modified CalB, which has allowed orders of magnitude increase in yields of protein. However, these modifications alter the quaternary structure of the protein. Here we produce soluble wild-type CalB in high yields in the cytoplasm of E.coli using a catalyzed system for cytoplasmic disulfide bond formation both in shake flasks and in fermentation in chemically defined media. The CalB produced had the expected stereospecific activity and had a higher activity than CalB from a commercial source.

Converting an Organic Light-Emitting Diode from Blue to White with Bragg Modes.

Organic light-emitting diodes (OLEDs) have been established as versatile light sources that allow for easy integration in large-area surfaces and flexible substrates. In addition, the low fabrication cost of OLEDs renders them particularly attractive as general lighting sources. Current methods for the fabrication of white-light OLEDs rely on the combination of multiple organic emitters and/or the incorporation of multiple cavity modes in a thick active medium. These architectures introduce formidable challenges in both device design and performance improvements, namely, the decrease of efficiency with increasing brightness (efficiency roll-off) and short operational lifetime. Here we demonstrate, for the first time, white-light generation in an OLED consisting of a sub-100 nm thick blue single-emissive layer coupled to the photonic Bragg modes of a dielectric distributed Bragg reflector (DBR). We show that the Bragg modes, although primarily located inside the DBR stack, can significantly overlap with the emissive layer, thus efficiently enhancing emission and outcoupling of photons at selected wavelengths across the entire visible light spectrum. Moreover, we show that color temperature can be tuned by the DBR parameters, offering great versatility in the optimization of white-light emission spectra.

Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding.

Oxidative protein folding in the ER is driven mainly by oxidases of the endoplasmic reticulum oxidoreductin 1 (Ero1) family. Their action is regulated to avoid cell stress, including hyperoxidation. Previously published regulatory mechanisms are based on the rearrangement of active site and regulatory disulfides. In this study, we identify two novel regulatory mechanisms. First, both human Ero1 isoforms exist in a dynamic mixed disulfide complex with protein disulfide isomerase, which involves cysteines (Cys166 in Ero1α and Cys165 in Ero1ß) that have previously been regarded as being nonfunctional. Second, our kinetic studies reveal that Ero1 not only has a high affinity for molecular oxygen as the terminal acceptor of electrons but also that there is a high cooperativity of binding (Hill coefficient >3). This allows Ero1 to maintain high activity under hypoxic conditions, without compromising cellular viability under hyper-hypoxic conditions. These data, together with novel mechanistic details of differences in activation between the two human Ero1 isoforms, provide important new insights into the catalytic cycle of human Ero1 and how they have been fine-tuned to operate at low oxygen concentrations.