An artificial protein cage made from a 12-membered ring

Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers

Optymization of synaptosome isolation methods and analysis of the proteome changes in murine brain induced by pilocarpine

Epilepsja jest chorobą neurologiczną o bardzo skomplikowanym i wciąż nie do końca poznanym podłożu. Jej najczęstszą odmianą jest padaczka skroniowa, występująca u ok. 40% pacjentów. Choroba ta charakteryzuje się nadmierną pobudliwością neuronów oraz reorganizacją architektury komórek nerwowych w mózgu w układzie limbicznym. Ze względu na olbrzymią rolę synaps w przekazywaniu sygnałów uzasadnione są badania tych struktur w celu wyjaśnienia mechanizmu epileptogenezy. Dobrym modelem synapsy są synaptosomy – struktury błonowe powstające podczas homogenizacji tkanki nerwowej. Struktury te zawierają błony pre- i postsynaptyczne, pęcherzyki wydzielnicze oraz mitochondria znajdujące się wcześniej w zakończeniach nerwowych. Synaptosomy zachowują funkcje synapsy, takie jak wydzielanie neurotransmiterów, produkcję ATP i utrzymanie potencjału błonowego.W niniejszej pracy przeprowadzono optymalizację izolacji synaptosomów w gradiencie Percollu, skupiając się głównie na zbadaniu wpływu czasu wirowania na skuteczność frakcjonowania homogenatu. Przydatność tej procedury porównano z techniką izolacji synaptosomów w gradiencie sacharozy. Skład białkowy uzyskanych frakcji analizowano przy pomocy spektrometrii mas (MS). Ze względu na niezadowalający stopień wzbogacenia frakcji synaptosomowej w białka synaptosomowe w przypadku procedury z Percollem w dalszej części pracy wykorzystano izolację w gradiencie sacharozy.W pracy użyto tkankę mózgu myszy, którym wcześniej została podana pilokarpina, związek wywołujący stan padaczkowy (SE) i wykorzystywany w jednym z modeli padaczki skroniowej (grupie kontrolnej – sól fizjologiczna). Mózgi badanych zwierząt pobrano w dwóch punktach czasowych: 24 godziny i 9 dni po podaniu pilokarpiny. Eksperyment miał na celu zbadanie zmian w profilu białkowym frakcji synaptosomowej wywołanych pilokarpiną. Białka synaptosomowe wyizolowane z półkul mózgowych badanych zwierząt rozdzielono z użyciem elektroforezy 2-wymiarowej, następnie otrzymane żele wybarwiono na białka fosforylowane oraz na wszystkie białka i zeskanowano. Obrazy żeli przygotowano do dalszej analizy poprzez odpowiednie przycięcie i usunięcie artefaktów.W trakcie pracy porównano również trzy sposoby przygotowania próbki do pomiarów MS techniką shot-gun oraz sprawdzono interferencję składników buforu z barwnikiem fluorescencyjnym Sypro Ruby, używanym do barwienia wszystkich białek w żelu.Epilepsy is a neurological disease with very complex and not well known etiology. 40% of epileptic patients show symptoms of temporal lobe epilepsy, one of the most common epilepsy types. Neural hyperexcitatory and architectonic changes of neurons in the limbic system are characteristic features of this disorder.Synapses play important role in the signal transduction processes, thus exploring their function in epileptogenesis seems to be justified. As synapses’ model synaptosomes can be used. Synaptosomes are membranous structures, which are formed during brain tissue homogenization. These structures contain pre- and postsynaptic membranes, synaptic viesicles and mitochondria derived from neuron endings. Synaptosomes mimic synapses functions, such as neurotransmitters release, ATP production and maintaining membrane potential. In the present study the optimization of a Percoll gradient procedure for synaptosomes preparation was performed. Optimization effort was focused on the adjustment of centrifugation time, which was the most important factor that influenced efficiency of the homogenate fractionation. Usefulness of the Percoll gradient procedure was compared with a sucrose gradient protocol. Protein composition of obtained fractions was analyzed by mass spectrometry (MS). However, Percoll fractions which were expected to contain synaptosomes were not sufficently enriched in proteins characteristic for synaptosomes. Due to this fact the sucrose gradient protocol was used in further experiments.In this work, the brain tissue of mice was used. Pilocarpine was applied to induce status epilepticus (SE) in one of the animal model of temporal lobe epilepsy. The brain hemispheres of examined animals were extracted after 24 hours (first group) and 9 days (second group) after pilocarpine administration (in control group saline was administered). The aim of the experiments was to investigate the changes in proteome of synaptosomes extracted from murine brain tissue. Synaptosomes’ proteins were separated by means of 2D electrophoresis, then obtained gels were stained for phosphoproteins and all proteins and subsequently scanned. Gel images were digitally pre-processed before further analysis.During these studies the three protocols for MS sample preparation in shot-gun experiments approach were compared and influence of buffer components on fluorescent gel staining with Sypro Ruby (used for staining all proteins) was checked as well

Artificial Protein Cage with Unusual Geometry and Regularly Embedded Gold Nanoparticles

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice

An ultra-stable gold-coordinated protein cage displaying reversible assembly

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery1, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein–protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging2,3. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein–protein interfaces. The addition of a gold (i)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S–Aui–S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(ii) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries