Development of a cysteinefree, covalent ligation strategy for the siteselective chemical modification of proteins

Bind&Bite‐ligation: Site‐selective protein ligation is a valuable tool in a variety of application such as pull‐downs, controlled surface‐immobilization and highly targeted affinity or fluorescence labeling. Yet, customizable cysteine‐free techniques that can be broadly applied especially for modification of proteins devoid of known ligands or structural information are sparse. This issue was addressed by introduction of a robust and straightforward two‐component labeling technique. It provides not only chemoselective but also spatial restriction of reactivity to its site of insertion in the protein sequence. Short peptidic recognition sequences form a stable heterodimeric coiled‐coil and serve as guiding moieties. The amine‐reactivity of the synthetic counterpart of the protein‐bound coiled‐coil peptide was hence confined solely to the protein region involved in coiledcoil assembly. In more detail, a highly modular coiled‐coil mediated lysine‐labeling technique was developed relying on a de novo designed heterodimeric coiled‐coil system. The acidic coiled‐coil peptide scaffold [1] with lysine to arginine substitutions (JB‐AK‐5(KR); sequence: EIAALEREIAALEREIAALER, Figure 1: red sequence) served as groundwork to attach desired labels (Figure 1: red star). The mutual site to be targeted was the unmodified basic coiled‐coil peptide [1] (JB‐AK‐6; sequence: KIAALKEKIAALKEKIAALKE), which was intended for incorporation in the sequence of the protein of interest (POI) (Figure 1: blue sequence). This approach was centered on in situ activation of the acidic coiled‐coil peptide by common watercompatible active‐ester chemistry (Figure 1: Bind&Bite‐Activation). The fast and selective protein modification then proceeds via molecular recognition and subsequent amino‐acylation (Figure 1: Bind&Bite‐ligation). Figure 1: Principle of the Bind&Bite‐Ligation. Covalent protein labeling achieved by: 1) preactivation of glutamate residues in acidic coiled‐coil peptide (red ribbon) modified with label of choice (red star); 2) addition of protein of interest (POI) in which the basic peptide sequence (blue ribbon) is incorporated; 3) coiled‐coil formation preorganizes activated carboxylates from the acidic and free amines from the basic coiled‐coil peptide and results in covalent stabilization of the coiled‐coil by isopeptide bond formation. Optimization of ligation conditions was performed using a specifically developed 2‐step streptavidinenhanced fluorescence polarization protocol. Treatment of the ligation mixture with guanidinium hydrochloride allowed to distinguish between covalent binding and non‐covalent coiled‐coil formation. In general best performance was achieved using the following standard procedure for Bind&Bite ‐ ligation: 1. Activation of the complementary peptide carrying the label: 5 / 100 μM peptide in EDC (aq., 500 mM) and NHS/sNHS (aq., 625 mM) for 30 min 2. Preparation of the protein of interest (POI) in desired buffer (PB, PBS, HEPES, etc…) 3. Dilution of activated peptide solution (1.) to working concentration with HPW 4. Incubation of POI with activated peptide solution (~ 10‐fold molar excess of labeling peptide) The ligation performance was further enhanced by replacing NHS as activating agent by its sulfonated analogue sulfoNHS (sNHS). In‐depth studies then revealed the required symmetrical minimum length 10 for both coiled‐coil peptides (16 AA for NHS, 14 AA for sNHS), as well as the possibility of purifying and storing the acidic coiled‐coil peptide in its activated form. Orthogonal Bind&Bite‐ligation: After establishing the general setup for Bind&Bite‐ligation, aromatic hydrophobic core variants were assessed as source of orthogonality for this approach. A basic peptide (protein‐bound) subjected to a full exchange of isoleucine and leucine residues to tryptophan was found to be most suitable (sequence: (KWAAWKE)3). Screening for a suitable acidic counterpart yielded the sequence: (ELys(TFA)AAWER)3. This pair of selected peptides, even when activated by sNHS ‐ exhibited a lag phase between desired (among each other) and undesired covalent binding (to the wild‐type Bind&Bite pair). The effect was one‐sided and not evident for the wild‐type activated acidic peptide, which modified either basic counterpart. Nevertheless this behavior might be exploited in a sequential one‐pot labeling procedure. The application range of the Bind&Bite‐technique could thereby be extended to labeling up to two proteins present in the same solution in quick succession. Positional encoded amine reactivity: The consequent evolution of the undirected approach was to achieve true positional encoding of amine reactivity. This was realized by the compatibility of selected amine‐reactive moieties with common SPPS conditions, enabling their incorporation in the acidic peptide during synthesis (Figure 2). The target peptide was adapted by exchange of all but one lysine residue with its isostere arginine. Figure 2: On‐resin introduction of miscellaneous ARSs from bis‐amine reactive crosslinker molecules. Introduction of ARS via Alloc‐protected surrogates (left), liberation of the amino group to be modified (middle, pink) and formation of mono amine‐reactive species (right) from treatment with homobifunctional crosslinkers (DFDNB, DSC, thioCDI, SADE; leaving groups marked in red) A comprehensive assessment of storage conditions and hydrolytic behavior revealed that the decay of even the most reactive peptides (NHS‐carbamates, ‐ esters) was still well outside the timeframe needed for sample preparation, liquid handling operations and finally coiled‐coil formation / ligation. The ranking order for tested reactive moieties by stability was determined as: DFDNB >> SCN > SADE >> E(NHS) ~ NHS A quantitative evaluation of ligation was performed on a limited panel of combinations by LC‐MS. Focusing mainly on suppression of centrically symmetrical off‐target effects, Dap residues were considered most promising for future introduction of amine‐reactive sites. Due to its Glu(NHS)‐ 12 bioisosteric properties and compact structure Dap was chosen as the surrogate of choice for full positional scans of peptides modified with varying amine‐reactive sites. The boundaries of orthogonal amine‐reactive sites were explored by screening positional scans of variants containing each of the proposed amine‐reactive residues. A binary labeling approach was discovered featuring high ligation turnover and strict orthogonality between the pairs of peptides. Quaternary orthogonal ligation (QOL): The most astonishing finding was the possibility of selectively ligating four individual heterodimers (Figure 3). Although a mix of 3 different activation strategies had to be applied and initial yields were rather low, 4 out of 6 lysine residues could be selectively addressed. The peptides acting as orthogonally matching pairs were are depicted below (Figure 3). Figure 3: Spatial illustration of all four orthogonal reactive heterodimers (QOL). Positional encoded orthogonality of aminereactive site (ARS) (red E) in acidic coiled‐coil peptide (red ribbon) and mutual reactive lysine (blue K) in basic coiled‐coil peptide (blue ribbon) (adapted from [1], image created by UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco) 13 Bind&Bite‐ligation applications: The versatility of the basic Bind&Bite approach in the context of modifying and immobilizing HIV‐1 envelope protein (Env) was elucidated. The integrity of this highly complex protein construct including its homotrimeric quaternary structure was preserved even after C‐terminal incorporation of the necessary basic coiled‐coil peptide separated via a 3 G4S unit (GGGGS) spacer. Analysis of antibody binding, gel shifts and covalent modification in fluorescence polarization assays provided further proof for the preservation of the protein structure, as well as the addressability of the Bind&Bite‐tag. Coiled‐coil directed ligation was found to proceed even for the highly diluted protein in cell supernatant. Hence, comprehensive experiments were conducted especially focusing on labeling under conditions highly adverse to common covalent ligations. Nevertheless, in situ activated coiled‐coil peptide resulted in reliable ligation in PBS, as well as in bacterial lysate and media containing up to 10 % FCS. Under similar conditions (10 % FCS), a site‐selective biotinylation enabled the selective immobilization of Env on streptavidin beads (pull‐down). Yet, the excess necessary for selective ligation, while maintaining minimum levels of unspecific modification was subject to fluctuations and would have to be subjected to further optimization. To circumvent this drawback, the amine‐reactive peptide was purified after activation with EDC/NHS, removing potentially problematic activating agents prior to Bind&Bite‐ligation. Although this setup displayed slightly decreased ligation performance overall, selective and background‐free labeling was possible for all tested conditions. The resulting fluorescently labeled product (fluoresceinylated Env‐6) was then utilized to distinguish between naive and Env‐specific PGT121 B cells by flow cytometry. Based on the intriguing synergy between the complex Env‐protein assembly and the Bind&Biteapproach, a novel amphipathic lipid anchoring technique was examined. It proofed fit to generate Bind&Bite‐ready liposomes that presented the acidic coiled‐coil peptide. The anchoring principle relied on the highly unfavorable translocation of a positively charged arginine across the liposomal membrane. This energetic barrier safeguarded the protein‐anchor assembly against mechanical “uprooting”. A discrete transmembrane length (53 Å) enabled near‐quantitative conjugation of Env. The resulting liposomes had diameter of approx. 300 nm and were characterized with a PDI of 0.11. Compared to conventional lipid anchors (cholesterol, bile acids) these Env‐coated liposomes exhibited superior stability and immobilization efficacy. In related works, coiled‐coil domains were mostly used to only be means to an end for site‐selective fluorescence labeling of proteins [2] [3]. By embracing the potential and inherent features of this versatile scaffold, over the course of the thesis the pure necessity of molecular recognition was made a virtue of. Straightforward activation of carboxylic acids in the acidic coiled‐coil peptide and the resulting siteselective amine ligation with its protein‐bound counterpart already paved the way for a variety of applications. These included controlled introduction of affinity and fluorescence tags in a delicate protein scaffold (HIV‐1 envelope protein, Env) under diluted (~ 700 pM) and highly crowded conditions (e.g. 10 % FCS / bacterial lysate). Combination of the same ligation strategy with a novel type of amphipathic membrane anchor enabled the covalent coating of liposomes with Env. The technique reliably ensured correct protein orientation, which is of paramount importance not only for the goal of HIV‐1 immunization. The advance of the basic methodology towards multiple layers of orthogonality and thus even more versatility was pursued by two approaches: Without changes to the activation procedure itself, solely by altering the nature of the hydrophobic core residues to aromatic structures an additional set of unidirectional orthogonal coiled‐coil peptides was developed. To achieve true positional encoded reactivity, sets of peptides featuring either single lysine or aminereactive residues were synthesized. Especially the incorporation of amine‐reactivity via various bivalent crosslinker molecules already during peptide synthesis can be seen as a landmark discovery. Comprehensive studies of the isolated single‐activated peptides under challenging storage and ligation conditions revealed the broad range of different stability profiles covered by the natures of the reactive residues. In combination with the corresponding target peptides presenting the free amine at different positions, these characteristics translated in distinct reaction fingerprints for each individual pair of peptides. In a first of its kind discovery, the coiled‐coil assembly provided fast and high yielding ligation of two pairs of coiled‐coil peptides or orthogonality between 4 heterodimers. This was achieved only by careful selection of amine‐reactive sites as well as placement of mutual reactive residues over the full length of the coiled‐coil. These intriguing results potentially open up new possibilities to utilize the true potential that coiledcoil peptides provide for directed ligation reactions. Thus, future work could be aimed at eliminating possible pitfalls like unwanted antipararallel hybridization. This problem could be solved by taking measures such as adjustment of ARS linker length or replacement of HCRs with Asn‐Asn pairs [4]. Perspectively, this could be the starting point to eliminate major drawbacks like the positional dependence of ligation yield and ‐speed as well as the centrically symmetric off‐target reactivity. With these problems resolved, a readily accessible and highly customizable toolkit for orthogonal protein labeling is within reach

Just transition toolbox for coal regions

As the worldwide remaining carbon budget decreases rapidly, countries across the globe are searching for solutions to limit greenhouse gas emissions. As the production and use of coal is among the most carbon-intensive processes, it is foreseeable that coal regions will be particularly affected by the consequences of a transformation towards a climate-neutral economy and energy system. Challenges arise in the area of energy production, environmental protection, but also for economic and social aspects in the transforming regions - often coined with the term "Just Transition". For the decision makers in coal regions, there is an urgent need for support tools that help to kick off measures to diversify the local economies while at the same time supporting the local workers and communities. The Wuppertal Institute aims to support coal regions worldwide by developing a Just Transition Toolbox, which illustrates the challenges and opportunities of a sustainable transition for a global audience. It comprises information about strategy development, sets recommendations for governance structures, fostering sustainable employment, highlights technology options and sheds light on the environmental rehabilitation and repurposing of coal-related sites and infrastructure. The toolbox builds on the work of the Wuppertal Institute for the EU Initiative for Coal Regions in Transition and takes into account country-specific findings from the SPIPA-partner countries India, Indonesia, South Africa, Japan, South Korea, Canada and the USA. The acronym SPIPA is short for "Strategic Partnerships for the Implementation of the Paris Agreement" an EU-BMU programme co-financed by the GIZ

Analysis of the historical structural change in the German hard coal mining Ruhr area (case study)

This case study examined the structural change in the Ruhr area caused by the low international competitiveness of German hard coal mining over the period from the late 1950s to 2015. It analysed the structural change process and the structural policies implemented as a reaction to this process with the objective to make this knowledge available for future structural change processes in other (coal) regions by deploying various qualitative and quantitative methods of empirical social and economic research. A discourse analysis helped to recognise who supported which structural policy approaches and why - and thus gives indications of the possible relevance of experiences for other regions

Analysis of the historical structural change in the German lignite mining area of Lusatia (case study)

This case study examined the structural change in Lusatia caused by the system change from a centrally planned economy to a market economy in the period 1990-2015. It analysed the structural change process and the structural policies implemented as a reaction to this process with the objective to make this knowledge available for future structural change processes in other (coal) regions by deploying various qualitative and quantitative methods of empirical social and economic research. A discourse analysis helped to recognise who supported which structural policy approaches and why - and thus gives indications of the possible relevance of experiences for other regions

Toolbox Transisi Berkeadilan untuk kawasan batu bara

Dengan semakin berkurangnya "anggaran karbon" atau carbon budget di seluruh dunia, berbagai negara sedang mencari solusi untuk mengurangi emisi gas rumah kaca. Karena produksi dan penggunaan batu bara dapat dikatakan sebagai salah satu penghasil emisi karbon yang sangat besar dan memicu perubahan iklim, oleh karena itu dapat diperkirakan bahwa wilayah penghasil batu bara akan sangat terdampak akibat transformasi energi dari sistem energi yang berbasis bahan bakar fosil menjadi energi terbarukan. Tantangan yang muncul tidak hanya di bidang produksi energi, perlindungan lingkungan, tetapi juga dalam aspek ekonomi dan sosial di kawasan kawasan batu bara yang tengah menghadapi transformasi - sering disebut dengan istilah "Transisi Berkeadilan". Para pengambil keputusan di wilayah penghasil batu bara, sangat membutuhkan alat pendukung untuk memulai langkah-langkah untuk mendiversifikasi ekonomi lokal yang disaat bersamaan juga mendukung pekerja dan masyarakat lokal. Transisi Berkeadilan ini membutuhkan perencanaan yang komprehensif, kebijakan baru dan penyesuaian serta keterlibatan semua pemangku kepentingan. Oleh karena itu, Wuppertal Institute merancang "Just Transition Toolbox" untuk memberikan dukungan bagi para praktisi di kawasan penghasil batu bara di seluruh dunia yang menggambarkan tantangan dan peluang dalam transisi berkelanjutan untuk audiens global. Toolbox Transisi Berkeadilan ini terdiri dari informasi tentang pengembangan strategi, rekomendasi untuk struktur tata kelola, mendorong lapangan kerja berkelanjutan, menunjukan pilihan teknologi dan menyoroti rehabilitasi lingkungan dan penggunaan kembali situs dan infrastruktur terkait batubara. Toolbox ini dikembangkan berdasarkan seperangkat alat yang dirancang oleh Wuppertal Institute melalui kerja sama dengan berbagai pemangku kepentingan atas inisiatif Uni Eropa untuk daerah-daerah kawasan Batubara yang berada dalam masa transisi. Toolbox ini juga menampilkan pelajaran yang diambil dari kawasan batu bara mitra SPIPA seperti India, Indonesia, Afrika Selatan, Jepang, Korea Selatan, Kanada, dan Amerika Serikat. Akronim SPIPA adalah kependekan dari "Kemitraan Strategis untuk Implementasi Perjanjian Persetujuan Paris" pada program UE-BMU yang dibiayai bersama oleh GIZ

Manual para una transición justa en regiones carboníferas

A medida que el presupuesto mundial de carbono disminuye rápidamente, los países de todo el mundo buscan soluciones para limitar las emisiones de gases de efecto invernadero. Dado que la producción y el uso del carbón son algunos de los procesos más intensivos en emisiones carbono, es previsible que las regiones que producen carbón se vean especialmente afectadas por las consecuencias de una transformación hacia la neutralidad climática. Estas regiones se enfrentan a retos en el ámbito de la producción de energía y la protección del medio ambiente, pero también a retos económicos y sociales, que se engloban en la necesidad de una "transición justa". Los responsables de la toma de decisiones en las regiones dependientes de la producción de carbón necesitan urgentemente herramientas de apoyo que ayuden a poner en marcha medidas para diversificar las economías locales y, al mismo tiempo, apoyar a los trabajadores y las comunidades locales. Wuppertal Institute busca apoyar a las regiones carboníferas de todo el mundo desarrollando una caja de herramientas para la transición justa, que ilustra los retos y las oportunidades de una transición sostenible para un público global. La caja de herramientas incluye recomendaciones para el desarrollo de estrategias y estructuras de gobernanza, líneas guía para la creación de empleo sostenible, el desarrollo de alternativas tecnológicas, la rehabilitación medioambiental y la reutilización de infraestructuras relacionados con el carbón. La caja de herramientas se basa parte del trabajo de Wuppertal Institute dentro de la Iniciativa de la UE para las Regiones Carboníferas en Transición y tiene en cuenta las circunstancias específicas de los países socios de la SPIPA: India, Indonesia, Sudáfrica, Japón, Corea del Sur, Canadá y Estados Unidos. El acrónimo SPIPA es la abreviatura de "Asociaciones Estratégicas para la Aplicación del Acuerdo de París", un programa UE-BMU cofinanciado por la GIZ

Analyse des historischen Strukturwandels im Ruhrgebiet (Fallstudie)

Diese Fallstudie untersuchte den durch die geringe Wettbewerbsfähigkeit des Steinkohlebergbaus ausgelösten Strukturwandel im Ruhrgebiet vom Ende der 1950er Jahre bis 2015. Mit Hilfe verschiedener qualitativer und quantitativer Methoden der empirischen Sozial- und Wirtschaftsforschung analysierte sie den Strukturwandelprozess und die in Reaktion auf diesen Prozess umgesetzte Strukturpolitik mit dem Ziel, dieses Wissen für zukünftige Strukturwandelprozesse in anderen (Kohle-)Regionen zur Verfügung zu stellen. Eine Diskursanalyse half zu erkennen, wer warum welche strukturpolitischen Ansätze unterstützte - und gibt damit Hinweise auf die mögliche Relevanz von Erfahrungen für andere Regionen

Analyse des historischen Strukturwandels in der Lausitz (Fallstudie)

Diese Fallstudie untersuchte den durch den Systemwechsel von der Plan- zur Marktwirtschaft ausgelösten Strukturwandel in der Lausitz im Zeitraum 1990-2015. Mit Hilfe verschiedener qualitativer und quantitativer Methoden der empirischen Sozial- und Wirtschaftsforschung analysierte sie den Strukturwandelprozess und die in Reaktion auf diesen Prozess umgesetzte Strukturpolitik mit dem Ziel, dieses Wissen für zukünftige Strukturwandelprozesse in anderen (Kohle-)Regionen zur Verfügung zu stellen. Eine Diskursanalyse half zu erkennen, wer warum welche strukturpolitischen Ansätze unterstützte - und gibt damit Hinweise auf die mögliche Relevanz von Erfahrungen für andere Regionen

Development of a Testing Funnel for Identification of Small-Molecule Modulators Targeting Secretin Receptors

The secretin receptor (SCTR), a prototypical class B G protein-coupled receptor (GPCR), exerts its effects mainly by activating Gαs proteins upon binding of its endogenous peptide ligand secretin. SCTRs can be found in a variety of tissues and organs across species, including the pancreas, stomach, liver, heart, lung, colon, kidney, and brain. Beyond that, modulation of SCTR-mediated signaling has therapeutic potential for the treatment of multiple diseases, such as heart failure, obesity, and diabetes. However, no ligands other than secretin and its peptide analogs have been described to regulate SCTRs, probably due to inherent challenges in family B GPCR drug discovery. Here we report creation of a testing funnel that allowed targeted detection of SCTR small-molecule activators. Pursuing the strategy to identify positive allosteric modulators (PAMs), we established a unique primary screening assay employing a mixture of three orthosteric stimulators that was compared in a screening campaign testing 12,000 small-molecule compounds. Beyond that, we developed a comprehensive set of secondary assays, such as a radiolabel-free target engagement assay and a NanoBiT (NanoLuc Binary Technology)-based approach to detect β-arrestin-2 recruitment, all feasible in a high-throughput environment as well as capable of profiling ligands and hits regarding their effect on binding and receptor function. This combination of methods enabled the discovery of five promising scaffolds, four of which have been validated and further characterized with respect to their allosteric activities. We propose that our results may serve as starting points for developing the first in vivo active small molecules targeting SCTRs

Bộ công cụ Chuyển dịch công bằng : cho các khu vực khai thác than

Do ngân sách các-bon còn lại trên toàn thế giới đang giảm nhanh chóng, các quốc gia trên toàn cầu đang tìm kiếm các giải pháp để hạn chế phát thải khí nhà kính. Ngành công nghiệp sản xuất và sử dụng than là một trong những ngành phát thải nhiều các-bon nhất, do vậy, các khu vực khai thác than sẽ bị ảnh hưởng đặc biệt bởi quá trình chuyển đổi sang hệ thống năng lượng và kinh tế trung hòa với khí hậu. Tại các khu vực thực hiện chuyển đổi, những thách thức không chỉ tồn tại trong lĩnh vực sản xuất năng lượng, bảo vệ môi trường, mà còn ở các lĩnh vực kinh tế và xã hội - thường được biết đến với khái niệm "Chuyển đổi Công bằng". Các cấp ra quyết định ở các khu vực khai thác than rất cần có các công cụ hỗ trợ giúp họ xác định các giải pháp chuyển đổi, vừa giúp đa dạng hóa nền kinh tế, vừa hỗ trợ người lao động và cộng đồng địa phương. Viện Wuppertal mong muốn hỗ trợ nâng cao năng lực cho các khu vực khai thác than trên toàn thế giới thông qua Bộ công cụ Chuyển dịch Công bằng – một tài liệu tổng quát, minh họa những thách thức và cơ hội của quá trình chuyển đổi bền vững. Bộ Công cụ này bao gồm kiến thức về xây dựng chiến lược, đưa ra các khuyến nghị về quản trị quá trình chuyển đổi, thúc đẩy việc làm bền vững, nêu bật các lựa chọn công nghệ và đề cập tới vấn đề phục hồi môi trường, tái sử dụng các cơ sở hạ tầng sản xuất than. Bộ Công cụ này được xây dựng dựa trên các nghiên cứu của Viện Wuppertal trong khuôn khổ “Sáng kiến của Liên Minh Châu Âu về chuyển dịch tại các khu vực khai thác than” và các kinh nghiệm thực tế của một số khu vực khai thác than trên toàn thế giới