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Strukturbestämning av proteiner är viktigt för att kunna förstå deras funktion och en snabbt utvecklande metod inom fältet är kryoelektronmikroskopi. Storleksbegränsningar förhindrar en bredare applikation av metoden eftersom små proteiner har för låg signal i förhållande till bakgrund för att kunna visualiseras som enstaka partiklar i elektronmiksoskopibilder. Hypotesen för projektet är att det är möjligt att avbilda väldigt små proteiner och kringgå den konventionella storleksbegränsningen genom att använda ett bärarprotein ((Putrescine Aminotransferase; YgjG) som kopplas till en affibody (Zwt) genom “helical fusion” och sedan binda ett litet målprotein till denna större struktur. Komplexet ska ge en tillräcklig storlek, symmetri och rigiditet för en lyckad elektronmikroskopi av bärare tillsammans med det lilla icke kovalent bundna målproteinet. För att karaktärisera den föreslagna bäraren, genomförs stabilitetstester genom CD, verifiering av inbindning av målproteinet i SPR, renhetsundersökning med SEC och slutligen kryoelektronmikroskopi för att testa konceptet. Det lilla målproteinet som kommer att avbildas i konceptstudien är en annan affibody (Z963), som i så fall skulle vara det minsta proteinet som någonsin har lösts med kryogenelektronmikroskopi. Resultaten visar att den undersökta tetramera-bäraren är väldigt stabil (Tm~ 85 oC) och kan tolerera en affibody-fusion med bibehållen bindning av multipla säten. Proteinet kan uttryckas rekombinant och renas till högt utbyte och bildar tetramerer även med fuserad affibody. De slutgiltiga resultaten från den kryoelektronmikroskopiska analysen inväntas fortfarande, men lovande griddar har skapats och en partikelselektion har gett klara 2-D klasser som också framhäver att det lilla målproteinet har bundit. Sammanfattningsvis har biofysikalisk karaktärisering indikerat att YgjG är en lovande bas för ett “imaging scaffold” och att preliminära enstaka-partikel kryoelektronmikroskopi analyser visar att den föreslagna strategin att undersöka små målproteiner är möjlig. / Determining structures of proteins is important to understand protein functions, and a rapidly evolving technique in this field is cryogen electron microscopy. However, size limitations are preventing wider applications of the technique because small proteins have poor signal to noise ratios and are not possible to distinguish in single-particle images. The hypothesis of this project is that it is possible to image very small proteins, bypassing the conventional size limitations of single-particle cryo-EM, by utilizing a carrier protein-scaffold (Putrescine Aminotransferase; YgjG) connected through helical fusion to an affibody (Zwt) that can bind to a small protein of interest. The complex provides a sufficient size, symmetry, and rigidity for successful electron microscopy also of the non-covalently bound small protein of interest. To characterise the proposed scaffold, thermal stability through CD, binding of target protein in SPR, purity through SEC and experiments towards proof-of-concept in cryo-EM will be performed. The small protein of interest to be imaged in the proof-of-concept setup is another affibody, called Z963, that would be the smallest protein ever solved with cryo-EM. The results show that the investigated tetrameric protein scaffold is a highly stable protein (Tm~85oC) that can tolerate affibody fusion with retained binding function of multiple sites. The protein can be recombinantly expressed and purified in high yield and forms tetramers also when fused to affibody. The cryo-EM results are still pending, but promising grids have been created and in an initial particle selection clear 2-D classes that also reveal the small bound protein of interest have been generated. To conclude, biophysical characterization indicates that YgjG is a promising base structure for an imaging scaffold and preliminary single-particle cryo-EM analyses show that the proposed strategy to investigate structures of small proteins of interest is feasible.

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Small proteins are difficult to study at high resolution with single-particle cryo-electron microscopy (cryo-EM). In general, sample properties such as large size (> 80 kDa), symmetry and rigidity are key to utilize this technology. To facilitate structural studies of small proteins as well, using cryo-EM, this project aims to incorporate a photo-inducible cross-link in a large and symmetric scaffold that is amenable for study, and covalently bind small proteins of interest to this scaffold. The scaffold in this project consists of rabbit muscle aldolase (157 kDa in tetrameric state) with an engineered affibody affinity protein (7 kDa) attached to the N-terminus of each aldolase monomer via a rigid helix fusion. The affibody-domain of the scaffold will be cross-linked to small proteins of structural interest, with a focus on a model target consisting of a second affibody with affinity for the affibody displayed on the aldolase scaffold. Photoconjugation of the affibody Zwt was performed to crosslink both the Fc of IgG and the anti-idiotypic affibody Z963, revealing that a methionine acceptor in the target is preferable but not necessary for UV crosslinking using BPA. Binding of affibodies rigidly displayed on of the scaffold to targets such as affibodies and antibody fragments was demonstrated , using surface plasmon resonance (SPR). / Att studera små protein vid hög upplösning med enpartikelsrekonstruktion i kryo-elektronmikroskopi (kryo-EM) är utmanande. Generellt så krävs stora (> 80 kDa), symmetriska och stabila protein för att använda sig av kryo-EM. Med målet att möjliggöra strukturbestämning och strukturella studier av små protein, så ska detta projekt föra in en foto-aktiverad korslänk i ett stort och symmetriskt bärarprotein. Bäraren består av aldolas från kaninmuskel (157 kDa som tetramer) med en affibody (7 kDa) kopplad till N-terminalen av varje aldolas-monomer via en rigidt fuserad helix. Affibody-domänen av bärarproteinet kan bilda korslänkar till små protein vars struktur sedan kan studeras. Fokus i projektet är ett modellprotein som består av en annan affibody som binder den affibody i bäraren. Fotokonjugering av affibodyn Zwt utfördes för att skapa korslänkar till både Fc av IgG, samt den anti-idiotypiska affibodyn Z963, vilket påvisade att en metionin-mottagare i målproteinet är fördelaktigt för UV korslänkning med BPA, men inte ett krav. Affinitet av affibodies i bärarproteinet till målprotein såsom andra affibodies och antikroppsfragment påvisades.

The breast cancer susceptibility protein, BRCA1, is a tumor suppressor that helps maintain genomic integrity. Changes in BRCA1 that effect DNA repair processes can fuel cancer induction. The Kelly lab, at the Virginia Tech Carilion Research Institute, has recently developed a new methodology that employs silicon nitride (SiN) microchips to isolate BRCA1 assemblies from the nuclear material of breast cancer cells. These microchips are coated with adaptor proteins that include antibodies against target proteins of interest. The adaptor proteins are added in sequential steps to the coated microchips, followed by an aliquot of sample containing the protein of interest, such as BRCA1. The Kelly lab, partnered with Protochips Inc., developed these devices as a robust, tunable platform to monitor molecular processes, and refer to them as 'Cryo-SiN' in cryo-Electron Microscopy (EM) imaging. We are currently using Cryo-SiN to recruit BRCA1 protein assemblies to the microchip surface under mild conditions, while simultaneously preparing them for cryogenic preservation and EM imaging. This strategy presents a viable alternative to antibody affinity columns that require stringent elution steps to obtain protein complexes from the column. Another advantage of the microchip strategy is that it requires only a 30-minute nuclear extraction, a 60-minute enrichment procedure, and a 5-minute microchip capture step--a total of 95 minutes from initially lysing the cells to plunge-freezing the EM specimens. Therefore, these novel approaches represent a major departure from classical separation procedures that often require days to complete, during which time active protein assemblies can readily dissociate or become inactive. Overall, our use of BRCA1-specific microchips may reveal changes in the BRCA1 architecture during various stages of cancer progression--a major gap in knowledge that persists in cancer research. / M. S. / Modern advances in the imaging technology used for cryogenic electron microscopy (cryo-EM) have offered researchers an extraordinary view into the world of biology at the nanoscale. Supplemental to these technical innovations is the development of tunable substrates based on functional new materials that revolutionize the sequestering of biological components from human cells, such as protein complexes formed in breast cancer cells. New developments of novel viewing substrates, given traditional electron microscopy viewing grids have remained unchanged for decades, is the logical next step into the future of enhanced cryo-EM imaging. Tunable microchip substrates, made using recently enhanced micro-engineering techniques, are currently under development for use in cryo-EM imaging. In this work I have examined these microchip substrates for their capacity to streamline the isolation of biomolecules such as the protein most prominently cited in breast cancer, known as the breast cancer susceptibility protein (BRCA1). Utilizing these novel microchip substrates in the Kelly Lab, I have collected and analyzed data containing BRCA1 proteins, formed in human breast cancer cells, toward the development of 3-dimensional protein structures that allow us to peer into the structure-function relationships of these proteins. New and exciting Cryo-EM data, collected using these newly developed microchips, has the potential to reveal obscure disease mechanisms being propagated at the molecular level in modern clinical practice, such as breast cancer.

<p>The fabrication of zone plate lenses that are used for focusing X-rays relies on nanofabrication techniques such as e-beam lithography, reactive ion etching, and electroplating. The circular grating-like zone plate pattern can have a smallest half-period, a so-called zone width, of down to 20 nm while it also needs to have a height that is 5 to 10 times the zone width to have good diffraction efficiency. This high aspect ratio structuring is a very challenging field of nanofabrication.</p><p>This diploma project has focused on improving the process step of fabricating the electroplating mold by cryo-cooling the polymer during the reactive ion etching with O₂. The low temperature causes passivation of the sidewalls of the mold during etching which results in a more ideal rectangular profile of the high aspect ratio plating mold.</p><p>By etching at -100 °C, structures with highly vertical sidewalls and no undercut were realized. The experiments showed that there is a tradeoff between the anisotropy of the zone profile and the formation rate of polymer residue, so-called RIE grass. Through a proper choice of process parameters the grass could be completely removed without introducing any undercut.</p> / QC 20100414

Les toxines de l’anthrax font partie de la famille des toxines A-B dans laquelle la moitié B se fixe à la membrane de la cellule permettant par la suite la translocation de la moitié A. Dans le cas de l’anthrax, la moitié B est représentée par le Protective Antigen (PA) et la moitié A par les deux protéines Edema Factor (EF) et Lethal Factor (LF). Après le recrutement par les récepteurs cellulaires (CMG2 et TEM8), PA s’organise en heptamère. Il peut fixer jusqu'à 3 ligands (EF et LF) avant d'être endocyté. Les modèles actuels de PA suggèrent que la baisse de pH à l’intérieur des endosomes permet un changement de conformation de la forme pré-pore vers la forme pore et que les ligands EF et LF passeraient au travers le pore pour entrer dans le cytoplasme. Cependant, le diamètre du pore est environ dix fois inférieur à celui des ligands (10 Å contre 100 Å). Un processus de folding/unfolding a été proposé mais demeure controversé. Afin d'identifier le processus de passage des facteurs EF et LF dans le cytoplasme, nous avons déterminé par cryo-microscopie électronique combinée avec l’analyse d’image les structures tridimensionnelles des complexes formés par PA et LF aux étapes prépore et pore. Par la suite, une étude complémentaire par dynamique moléculaire nous a permis de modéliser à haute résolution les différentes interactions qui ont lieu au sein du complexe. La structure 3D du complexe prépore combiné à 3 LF a été déterminée à une résolution de 14 Å. Nous avons aussi calculé une structure préliminaire du complexe pore également combiné à 3 LF Celles-ci n’ont jamais été résolues auparavant et leur connaissance permet d’envisager l’étude en profondeur du mécanisme infectieux de l’Anthrax in vivo. / The anthrax toxins are part of the A-B toxin family in which the B moiety binds to the cell membrane allowing subsequent translocation of the A moiety. In the case of anthrax, the B moiety consists of the Protective Antigen (PA), and the A moiety is composed of the two proteins Edema Factor (EF) and the Lethal Factor (LF). After being recruited by the cell receptors (CGM2 or TEM8), PA organizes itself into a heptamer. It can bind up to three ligands (either EF or LF) before being endocytosed. Current models suggest that the decrease of pH inside the endosomes allows a conformational change of PA from a prepore form to a pore form that allows the EF and LF ligands to pass through the pore and enter the cytoplasm. However, the pore diameter is about ten times smaller than the diameter of the ligands (10Å versus 100Å). A process of ligand folding / unfolding has been proposed, but remains controversial. To identify the mechanism by which EF and LF enter the cytoplasm, we have used cryo-electron microscopy and three-dimensional image analysis to determine the 3D structure of the PA-LF complexes in the pre-pore and pore conformations. Then, we used molecular dynamics to modelise at high resolution the different interactions that occur within the complex. The 3D structure of the pre-pore complex bound with three LF ligands has been determined at 14Å resolution. We also calculated a preliminary structure of the LF-bound pore complex. These structures have never been reported before. They provide the necessary information to study in depth the mechanism of anthrax infection in vivo.