Project description:Protein–protein interactions (PPIs) are shaped by evolutionary pressures that fine-tune binding affinities and drive the epistatic relationships that support functional outcomes. Here, we used the complex of bovine pancreatic trypsin inhibitor (BPTI) and chymotrypsin as a model system to study how mutations at one or two positions affect binding affinity and epistasis. To predict the binding affinity landscape of the BPTI–chymotrypsin complex, we combined deep sequencing data, obtained from a saturation scanning mutagenesis BPTI library, with a machine-learning (ML) model. Using this ML model, which was trained on a subset of experimental binding data, we predicted the binding affinities and epistatic interactions across thousands of single and double BPTI mutants, including those not observed in the library. Our predictive approach completed missing data points and enabled us to reveal global trends in affinity changes and mutation couplings within specific binding interface positions. Our analysis revealed that different mutations in the same position may have different effects on affinity, with most double mutations leading to increased epistasis, particularly at hotspot positions, thereby indicating a cooperative binding effect. In most cases, affinity and epistasis were inversely correlated, with affinity enhancement of double-mutant variants being associated with negative epistasis. Our approach can be readily generalized to predict mutation effects in larger combinatorial libraries and in proteins for which structural information is lacking.
Project description:Native metabolomics method validation for chymotrypsin.
1. Limit of detection Mollasamide- Chymotrypsin
2. Flowinjection of Mollasamide over UHPLC gradients (with make-up).
3. Binding tests with chymotrypsin and different standards.
Project description:1) Analysis of HTPS FT (analysis of the background, without protease) 2) Analysis of specificity and activity of Thrombin and Chymotrypsin at multiple time point (0,5,15,30,60,120,240 min) 3) Analysis of specificity and activity of Thrombin and Chymotrpysin with 2 hr incubation at 20C, 25C and 37C
Project description:To investigate the global molecular mechanisms underlying the phenotype induced by ZZEF1 loss in pancreatic β-cells and subsequent treatment with azoramide, we isolated pancreatic islets from β-cell-specific ZZEF1 knockout mice (βZKO-Mip) and their littermate controls. Both groups were fed a HFD for four weeks and treated with either azoramide (10 µM) or DMSO (10 µM) for 24 hours.
Project description:Native metabolomics method validation for chymotrypsin.
1. Limit of detection Mollasamide- Chymotrypsin
2. Flowinjection of Mollasamide over UHPLC gradients (with make-up).
3. Binding tests with chymotrypsin and different standards.
Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases Transcriptional response to protease treatments (kallikrein, papain and chymotrypsin) of Bifidobacterium breve 210B
Project description:Myeloid cell lines (K562 and HEL) were treated overnight with CHIR99021 or a vehicle control (DMSO). CHIR99021 treatment inhibits GSK3B within the destruction complex functioning in Wnt/beta-catenin signalling pathway, thereby preventing beta-catenin degradation and promoting its stabilization. Following the overnight incubation, beta-catenin RIP (RNA immunoprecipitation) was performed in both K562 and HEL cells. RNA samples obtained from beta-catenin RIP in these cells were then sequenced to identify beta-catenin-associated RNAs under CHIR99021 treatment compared to basal conditions (DMSO control).
Project description:mESCs were cultured at least 2 weeks in medium medium: N2B27 for SILAC (DMEM/F12 for SILAC (AthenaES), Neurobasal for SILAC (AthenaES), Sodium Pyruvate (40 mg/mL), N2 (1X), B27 (0.5X), Pen/Strep (1%), L-glutamine (2 mM), beta-mercaptoethanol (50 µM)) + 2i/LIF supplemented with (medium) 13C6 15N4 L-arginine (0.65 mM), (medium) 13C6 L-arginine (0.55 mM), and L-Proline (200 mg/L)84. Before switching from light to heavy medium, cells were treated with 0.05 µg/mL CHX or equivalent dilution of DMSO in 6-well plates. We treated the cells with 0.05 µg/mL CHX. At timepoint 0 h, we replaced medium medium with pre-warmed heavy medium: N2B27 for SILAC (Thermo scientific) supplemented with (heavy) 13C6 15N2 L-Lysine-2HCl (0.65 mM), (heavy) 13C6 15N4 L-Arginine-HCl (0.55 mM), and L-Proline (200 mg/L) for mESC.