Delivery of expression cassettes into mammal cell lines with bacteriophage vector

Laccase-like proteins are multicopper oxidases involved in several biological and industrial processes. Their application is commonly limited due to inhibition by fluoride and chloride, and as-isolated proteins are often substantially activated by heat, suggesting that multiple redox states can complicate characterization. Understanding these processes at the molecular level is thus desirable but theoretically unexplored. This paper reports systematic calculations of geometries, reorganization energies, and ionization energies for all partly oxidized states of the trinuclear copper clusters in realistic models with ∼200 atoms. Corrections for scalar-relativistic effects, dispersion, and thermal effects were estimated. Fluoride, chloride, hydroxide, or water was bound to the T2 copper site of the oxidized resting state, and the peroxo intermediate was also computed for reference. Antiferromagnetic coupling, assigned oxidation states, and general structures were consistent with known spectroscopic data. The computations show that (i) ligands bound to the T2 site substantially increase the reorganization energy of the second reduction of the resting state and reduce the redox potentials, providing a possible mechanism for inhibition; (ii) the reorganization energy is particularly large for F^– but also high for Cl^–, consistent with the experimental tendency of inhibition; (iii) reduction leads to release of Cl^– from the T2 site, suggesting a mechanism for heat/reduction activation of laccases by dissociation of inhibiting halides or hydroxide from T2

Presentation of protein domains on the scaffold of coiled-coil protein origami tetrahedron

Glavna prednost polipeptidih nanostruktur, v primerjavi z večino ostalih polimerov, je tvorba zelo kompleksnih struktur, ki jih določa zaporedje aminokislin. Narava je tekom evolucije raziskala le majhen del teoretično možnih proteinskih zvitij, medtem ko večji del t.i. proteinskega vesolja ostaja neraziskan. Pristopov k razvoju in raziskovanju proteinskih zvitij, ki v naravni niso prisotna, je več. Eden od pristopov k racionalnemu načrtovanju de novo proteinskih zvitij je proteinski origami na osnovi ovitih vijačnic. Proteinski origami iz ovitih vijačnic je načrtovan po principu modularnosti in daljnosežnih interakcij med segmenti ovitih vijačnic. Za to proteinsko zvitje so značilne raznolike poliedrske kletke, pri katerih stranice tvorijo ovite vijačnice, ki so med seboj povezane z gibljivimi proteinskimi povezovalci in s hidrofilno votlino na sredini nanokletke. V doktorskem delu smo raziskali strategije predstavitve proteinskih domen na ogliščih tetraedrične nanokletke z direktno genetsko fuzijo. Delovanje razvitega sistema smo preizkusili s predstavitvijo ene (GFP), dveh (GFP in RFP) ali štirih (štiri GFP ali RFP) proteinskih domen. Na podlagi in silico modeliranja in analize sipanja rentgenskih žarkov pri nizkih kotih se je pokazalo, da je proteinska domena, predstavljena na četrtem oglišču kot fuzija na C-koncu verige, gibljiva. Z zamenjavo mesta fuzije znotraj zaporedja TET12SN smo jo prestavili v notranjost polipeptidne verige in tako zmanjšali njeno gibljivost. Po učvrstitvi vseh štirih domen smo pripravili serijo sedmih konstruktov z različnimi razmerji GFP in RFP, pri katerih smo dokazali uspešno predstavitev proteinskih domen z analitsko gelsko filtracijo in s sipanjem rentgenskih žarkov pri nizkih kotih. TET12SN-RRRR, tetraedrično proteinsko ogrodje, dekoriranim s štirimi RFP proteinskimi domenami, smo uspešno uporabili kot cepivo za predstavitev antigenov. Miši, imunizirane s TET12SN-RRRR so razvila IgG protitelesa proti antigenu RFP hitreje in bolj zanesljivo kot miši, ki so bile imunizirane z monomernim antigenom. Kot alternativno strategijo predstavljanja proteinskih domen na proteinskem origamiju smo TET12SN dekorirali s SpyCatcher/SpyTag proteinskimi domenami, katere smo uporabili za izgradnjo centralnega tetraedra, dekoriranega z (i) enim, (ii) dvema ali (iii) tremi ligandi vključenimi v tetraedrične proteinske kletke. Trenutno sta velikost in kompleksnost poliedričnih proteinskih origamijev omejena z številom dostopnih ortiginalnih ovitih vijačnic. Da bi se izognili tej omejitvi, smo raziskali če lahko uspešno načrtujemo tetraedričen proteinski origami, pri katerem isti par ovitih vijačnic uporabimo večkrat. Dokazali smo, da isti par ovitih vijačnic znotraj tetraedrične nanokletke lahko uporabimo največ dvakrat, ter na tak način uspešno načrtovali in okarakterizirali tetraedrično nanokletko s ponovljenimi pari ovitih vijačnic enkrat (TET12SN(2CC)), dvakrat (TET12SN(22CC)) in trikrat (TET12SN(222CC)). Da bi dokazali modularnost razvitega sistema predstavljanja proteinskih domen na ogliščih, smo pripravili serijo sedmih konstruktov z različnimi razmerji GFP in RFP, le da smo za osnovno ogrodje uporabili TET12SN(22CC). Uspešno izolirane proteine iz te serije smo okarakterizirali z analitsko gelsko filtracijo in sipanjem rentgenskih žarkov pri nizkih kotih, medtem ko smo konstrukt dekoriran z štirimi rdečimi fluorescentnimi proteini še dodatno vizualizirali z krio-elektronsko mikroskopijo. Predstavljen sistem predstavljanja proteinskih domen na ogliščih proteinskih origamijev ima potencial za uporabo v medicinske, industrijske in biotehnološke namene.Compared to other biopolymers, polypeptide nanostructures are able to form highly complex structures that are defined by the aminoacid sequence. Nature explored only a small portion of all protein folds, while huge regions of \u27protein space\u27 remain unexplored. There are several approaches to discovering or creating new protein folds – coiled-coil protein origami (CCPO) is one of such. CCPO is de novo rationally designed protein fold from linked coiled-coil (CC) forming modules. CCPO is modular, composed of CCs that form the edges around the internal hydrophilic cavity and can form diverse polyhaedral shapes. In the presented thesis we explored the strategies of presenting protein domains at the vertices of tetrahedral CCPOs by direct genetic fusion. As a proof of principle, four tetrahedral CCPO (TET12SN) constructs were decorated either with one green fluorescent protein (GFP), one green and one red fluorescent protein (RFP) or four GFPs or four RFPs. All constructs expressed well and were characterised by small-angle X-ray scattering (SAXS), which, coupled with in silico modelling, revealed that there was some undesired flexibility of the fourth presented protein domain. The mobility was reduced by modifying the insertion site – and to further explore the developed system, seven additional constructs, with different ratios of GFP : RFP were prepared and analysed with SAXS and analytical gel filtration. Moreover, TET12SN-RRRR, tetrahedral CCPO decorated with four RFPs, was used as a vaccine for antigen presentation. Mice immunised with an antigen, presented on a scaffold (TET12SN-RRRR), developed IgG antibodies against RFP faster and with higher titer compared to mice immunised with a monomeric antigen. As an alternative strategy of protein domain grafting onto the CCPO scaffold, TET12SN was decorated with SpyCatcher/SpyTag protein domains, that were used to create bigger protein complexes of a tetrahedron decorated with (i) one, (ii) two or (iii) three additional ligand tetrahedral CCPO. Currently, the complexity of polyhaedral CCPOs are limited by the number of available orthogonal CCs. We investigated if the same CC pair could be used several times within the same structure. We successfully designed and characterised a tetrahedral CCPO with one (TET12SN(2CC)), two (TET12SN(22CC)) or three (TET12SN(222CC)) CC pair that occured two times in the sequence. In order to prove that protein domains could still be presented on CCPO with repeated CC pairs, seven protein constructs with varying ratios of GFP : RFP were created utilizing newly designed CCPO TET12SN(22CC). All successfully isolated proteins from the last series were also characterized by an analytical gel filtration and SAXSmoreover, protein construct with four RFPs was characterised in more detail with cryoelectron microscopy. The developed system for presenting protein domains at the vertices of tetrahedral CCPO offers a powerful tool in expanding toolkit of protein origami, that could one day be used in medical, industrial and/or biotechnological applications

Structural polymorphism of coiled-coils from the stalk domain of SARS-CoV-2 spike protein

Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N-terminal segment of the stalk does not form coiled-coils and remains disordered in solution. The C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of the full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with an unusual combination of a-d and e-a-d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled-coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS-CoV-2 stalk sequence

Metal ion–regulated assembly of designed modular protein cages

Coiled-coil (CC) dimers are versatile, customizable building modules for the design of diverse protein architectures unknown in nature. Incorporation of dynamic self-assembly, regulated by a selected chemical signal, represents an important challenge in the construction of functional polypeptide nanostructures. Here, we engineered metal binding sites to render an orthogonal set of CC heterodimers Zn(II)-responsive as a generally applicable principle. The designed peptides assemble into CC heterodimers only in the presence of Zn(II) ions, reversibly dissociate by metal ion sequestration, and additionally act as pH switches, with low pH triggering disassembly. The developed Zn(II)-responsive CC set is used to construct programmable folding of CC-based nanostructures, from protein triangles to a two-chain bipyramidal protein cage that closes and opens depending on the metal ion. This demonstrates that dynamic self-assembly can be designed into CC-based protein cages by incorporation of metal ion–responsive CC building modules that act as conformational switches and that could also be used in other contexts

Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials