Thesis (Ph.D.)--McMaster University (Canada), 1995. / Source: Dissertation Abstracts International, Volume: 56-12, Section: B, page: 6729. Adviser: J. P. Capone.

Thesis (Ph.D.)--McMaster University (Canada), 1995. / Source: Dissertation Abstracts International, Volume: 56-12, Section: B, page: 6730. Adviser: J. P. Capone.

Proteins can be labeled with different tags to enable their structural and functional investigations. In addition, labeling proteins at specific sites helps in studying the conformational dynamics of these molecules. A plethora of methods is available to facilitate labeling, choice of which largely depends on the requirements and the anticipated end results. In general, the various labeling methods can be classified into four different classes based on the stage at which labeling is performed, namely post translational labeling, non-ribosomal synthesis, in vitro translation and in vivo translation. Interestingly all these techniques use different unnatural amino acids for this purpose. Protein folding is one among the many applications that requires tailoring proteins with special molecules or labels for deducing the folding pathway. Understanding the protein folding problem is a key for answering questions concerning protein behavior and thus, will provide strategies to solve protein misfolding diseases. Protein folding is one among the unsolved problems in biology and in particular understanding the in vivo behavior of proteins in the complex cytoplasm environment with a cellular density of approximately 350 to 400 mg/ml is more critical. It is evident that there is a difference in the behavior and folding of proteins in vivo and in vitro and to deduce more insights in this aspect the protein of interest is to be labeled with a sensitive probe. The in vivo translation method offers a good method of choice for labeling the protein at a specific position and monitoring its behavior. To study the ultimate goals of acquiring knowledge of the in vivo behavior and folding characteristics of proteins, the first step of establishing an efficient labeling technique is quintessential and as a starting step, this project aims to label a β-clam protein, cellular retinoic acid binding protein I (CRABP I) a 136 amino acid protein, with a sensitive unnatural fluorescent amino acid probe in vivo in E. coli cells.

Natrium-kaliumpumpen spelar en väsentlig roll i en rad fysiologiska funktioner då den upprätthåller den elektrokemiska gradienten över cellmembranet. Ytterligare så är störningar i dess funktion associerade med flera neurologiska sjukdomar. Proteinet är en heterodimer av α– och β–subenheter, ibland även associerat med en tredje γ (FXYD) subenhet, vilket gör det problematiskt att studera dess högre ordningens organisation i cellmembranet med hjälp av konventionella, relativt storskaliga inmärkiningsprober såsom antikroppar. Inkorporering av icke-kanoniska aminosyror är ett nyutvecklat och växande område som erbjuder en lösning. Genom CuAAC– och SPIEDAC–klickkonjugationsreaktioner kan organiska färgämnen (fluoroforer) snabbt och specifikt fästas i sidokedjor med motsvarande reaktiva grupper på jonpumpen, vilket skapar en liten och icke-invasiv inmärkningsprob för fluorescensmikroskopi. För att specifikt studera alla tre subenheter samtidigt krävs inmärkning med tre olika fluoroforer). Syftet med detta projekt var att lyckas med trefärgsinmärkning genom inkorporering av icke-kanoniska aminosyror, och därigenom underlätta studerandet av hur natrium-kaliumpumpens subenheter ordnar sig i cellmembranet. Transient transfekterade HEK293T-celler med membraninmärkta jonpumpar studerades med hjälp av fluorescensmikroskopi, vilket kompletterades med gelfluorescensavbildning och immunoblotting. Samtidigt gjordes proteinuttryck och tvåfärgsinmärkning av alla nonsenskodonmuterade subenheter i kombination med varandra och var synlig i proteingel, där endast α och β tidigare hade samuttryckts. α/γ parinmärkning visade sig framgångsrik när de samtransfekterades med β av vildtyp. En autofluorescenseffekt i en av färgkanalerna påverkade resultaten för mikroskopin. Trefärgsinmärkning observerades inte i gelen, och uttrycket av subenheterna (varav α var ersatt för detta experiment) var i stort sett obefintligt. Otydlighet består därmed huruvida trefärgsinmärkning eller trippelsamuttryck är möjligt med de bioortogonala translationssystemen som användes i detta projekt på jonpumpen. / Na+/K+-ATPase is an essential ion pump protein in a host of physiological functions as it maintains the electrochemical gradient across cell membranes. Additionally, its dysfunction is implicated in several neurological diseases. The protein is a heterodimer of α and β subunits, occasionally associated with a third γ (FXYD) subunit, which makes studying its higher order organization in the cell membrane difficult using conventional, relatively large scale labeling probes such as antibodies. Non-canonical amino acid incorporation is an emerging field which offers a solution. Via CuAAC and SPIEDAC click conjugation reactions, organic fluorophores can be specifically attached to the side chains of residues of the ion pump with corresponding reactive moieties, creating a small and noninvasive probe for fluorescence microscopy imaging. In order to specifically image all three subunits concurrently, three color labeling is required. The objective of this project was to achieve three color labeling via non-canonical amino acid incorporation to aid in the study of the cell membrane localization of the subunits of Na+/K+-ATPase. Fluorescence microscopy of transiently transfected and live cell labeled HEK293T cells was complemented by in gel fluorescence imaging and immunoblotting. Coexpression and two color labeling of all nonsense codon subunit mutants in combination was shown in gel, of which only α and β had previously been coexpressed. α/γ dual labeling proved successful when cotransfected with wild type β. An autofluorescent effect in one of the color channels compromised the microscopy results. Three color labeling was not observed in gel, and expression of the subunits (including a substitute for α) was middling to absent. It remains unclear whether three color labeling or triple coexpression is a possibility with the bioorthogonal translation systems used in this project.