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Avidity is an effective and frequent phenomenon employed by nature to achieve extremely high-affinity interactions. As more drug discovery efforts aim to disrupt protein-protein interactions, it is becoming increasingly common to encounter systems that utilize avidity effects and to study these systems using surface-based technologies, such as surface plasmon resonance (SPR) or biolayer interferometry. However, heterogeneity introduced from multivalent binding interactions complicates theanalysis of the resulting sensorgram. A frequently applied practice is to fit the data based on a 1:1 binding model, and if the fit does not describe the data adequately, then the experimental setup is changed to favor a 1:1 binding interaction. This reductionistic approach is informative but not always biologically relevant. Therefore, we aimed to develop an SPR-based assay that would reduce the heterogeneity to enable the determination of the kinetic rate constants for multivalent binding interactions using the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human receptor angiotensin-converting enzyme 2 (ACE2) as a model system. We employed a combinatorial approach to generate a sensor surface that could distinguish between monovalent and multivalent interactions. Using advanced data analysis algorithms to analyze the resulting sensorgrams, we found that controlling the surface heterogeneity enabled the deconvolution of theavidity-induced affinity enhancement for the SARS-CoV-2 spike protein and ACE2 interaction.

Supported lipid bilayers (SLBs) serve important roles as minimalistic models of cellular membranes in multiple diagnostic and pharmaceutical applications as well as in the strive to gain fundamental insights about their complex biological function. To further expand the utility of SLBs, there is a need to go beyond simple lipid compositions to thereby better mimic the complexity of native cell membranes, while simultaneously retaining their compatibility with a versatile range of analytical platforms. To meet this demand, we have in this work explored SLB formation on PEDOT:PSS/silica nanoparticle composite films and mesoporous silica films, both capable of transporting ions to an underlying conducting PEDOT:PSS film. The SLB formation process was evaluated by using the quartz crystal microbalance with dissipation (QCM-D) monitoring, total internal reflection fluorescence (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) for membranes made of pure synthetic lipids with or without the reconstituted membrane protein β-secretase 1 (BACE1) as well as cell-derived native lipid vesicles containing overexpressed BACE1. The mesoporous silica thin film was superior to the PEDOT:PSS/silica nanoparticle composite, providing successful formation of bilayers with high lateral mobility and low defect density even for the most complex native cell membranes., Funding: Swedish Foundation for Strategic Research (SSF)Swedish Foundation for Strategic Research [RMA11-0104]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; SSFSwedish Foundation for Strategic Research [RIT15-0119]; Onnesjo Foundation

Detta självständiga arbete har syftet att undersöka hur medlemmar i RFSL under tidsperioden1972 – 1980 såg på föreställningar om manligt och kvinnligt och om det verkligen skedde enreformation inom RFSL som tidigare forskning påpekade. För att besvara detta syfte har enkvalitativ textanalys använts för att analysera de officiella RFSL tidskrifterna RFSL – Nytt ochTidskrift för RFSL. Arbetet har använt begreppen hegemonisk maskulinitet och genusteori föratt se hur föreställningar om manligt och kvinnligt kom till uttryck inom de nämndatidskrifterna. Resultatet påvisar att trotts det har skett en reformation av RFSL finns detfortfarande tydliga föreställningar om vad som anses vara manligt och kvinnligt. Detta görstydligt utifrån ett genusperspektiv där tidskriften vid flera tillfällen väljer att associera kvinnamed negativa egenskaper medan mannen associeras med positiva egenskaper. Därutöver görtidskrifterna tydliga val att ge plats åt män i artiklar och exkluderar kvinnor från de artiklarsom publiceras. Utifrån begreppet hegemonisk maskulinitet klargörs det att de som har dendominerande makten inom RFSL är män och dessa homosexuella män väljer att underordnasig rådande normer gällande maskulinitet med avsikten att ta del av dess fördelar, men ävensom ett medel att vidare försämra kvinnans position. Genom att männen inom RFSLunderkastar sig samhällets normer om manligt och kvinnligt kan de trots sin sexuella läggningta del av maskulinitetens fördelar. Arbetet påvisar att det till en viss del existerar tydliganormer inom organisationen RFSL som männen väljer att följa och genom att anamma dessanormer missgynnas de kvinnor som är aktiva inom organisationen. Utöver detta påvisarresultatet av arbetet att ämnet har en didaktisk koppling till skolans värld. Då tidskrifterna kananvändas som ett medel för eleverna att utveckla deras källkritiska vid granskning av ettkällmaterial.

The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding.

Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr24 and Ser256 in FOXO1 to stimulate binding of 14-3-3 proteins, causingFOXO1inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser383 and Thr649 in FOXO1. In this study, we identified Ser22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser22 phosphorylation impairs Thr24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser383 and Thr649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.