In the course of this Ph.D. thesis, new noncanonical amino acids were genetically encoded to study the interactions and structure of proteins.
In the first project, a furan-based photocrosslinking chemistry was successfully added to the genetic code of E. coli and mammalian cells. Therefore, a furan-derived lysine analogue was synthesized and a new orthogonal pyrrolysyl-tRNA-synthetase/tRNAPyl pair was evolved. A published selection system was therefor established and selection parameters were optimized. Additionally, several PylRS libraries were created for directed evolution experiments. The co-translational incorporation at user-defined sites in proteins with a furan-based noncanonical amino could be demonstrated with high efficiency and fidelity. For the first time, protein-RNA crosslinking experiments could be conducted with red light, using a furan-modified protein, and methylene blue as a photosensitizer in vitro. The crosslinking properties were studied in detail, using a medical relevant RNA-protein complex (TAR-Tat complex of HIV-1). The arginine-rich motif (ARM) of the HIV-1 Tat protein was fused N-terminally to GFP and the binding affinity and crosslinking behavior towards TAR-RNA were studied. It was demonstrated that the formation of the crosslinking product is dependent on the spatial orientation of the furan-derived noncanonical amino acid within the protein, complex formation and presence of singlet oxygen. The application of red light offers high penetration depth in biological samples such as tissues, and obviates the need for UV-light controlled, traditional photochemistries. This technique might be applicable for the development of DNA and RNA-targeting biologics, featuring a red-light controlled chemical warhead. This approach could be interesting for the application in peptide therapeutics especially in photodynamic therapy. Furthermore, the site-specific incorporation of furan-based noncanonical amino acids in response to the amber stop codon could be demonstrated in mammalian cell culture. Future applications could be focused on the in vivo detection of transient protein-RNA or DNA interactions in live bacterial or mammalian cells. In efforts to circumvent the supplementation of photosensitizers, the application of genetically encoded photosensitizing proteins could be of potential interest.[210] Moreover, furan-mediated biorthogonal labeling could be transferred to proteins.[98] In addition, the structural basis of the evolved, polyspecific PylRS was investigated by X-ray crystallography in collaboration with Anne-Marie Weber (AG Welte). The mode of amino acid recognition and the polyspecificity of PylRS_AF (harboring mutations Y306A and Y384F) could be illuminated in detail. The influence of the mutations on the shape and constitution of the binding pocket could be explained and novel amino acid residues at the rear end of the new, extended binding pocket could
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be identified. These amino acid residues are potential targets to further enhance binding capacity of the binding pocket in the future.
In a second project, the genetically encoded biosynthesis of spin-labeled proteins in E. coli could be demonstrated, giving new perspectives in precisely measuring structural information of proteins. This direct method of protein spin-labeling obviates the need for chemical labeling steps and cysteine mutagenesis as required for traditional spin-labeling based e.g. on sulfhydryl-reactive labeling reagents. By employing the potentially stable hydroxylamine surrogates of the ncAA of interest in in vivo selection experiments, a new orthogonal PylRS/tRNAPyl pair was identified by directed evolution and genetic selection. The evolved PylRS-SL1 exhibits crossreactivity towards its paramagnetic nitroxide amino acid analogue. The site-specific incorporation of spin-labeled amino acids could be demonstrated at user-defined positions in proteins in response to the amber stop codon. The integrity degree of spin-labeled proteins was investigated and 50 - 70 % could be obtained for singly-labeled GFP mutants. Nitroxide free radicals were shown to be unexpectedly more stable in bacteria compared to earlier reported stabilities in eukaryotes.[203, 211-212] The spectroscopic properties of the spin-labeled amino acid were investigated in collaboration with the AG Drescher. In DEER distance measurements of doubly spin-labeled proteins, the spectroscopic value of this method was thoroughly assessed. Therefore, the structure of bacterial thioredoxin, bearing two essential cysteines, was studied. Thioredoxin cannot be studied in its native form using classical MTS spin-labeling techniques without impairing on the catalytic activity by mutagenesis and additionally forcing the protein in a reduced-like conformation. The performance of the spin-labeled amino acid was investigated to resolve precise distance distributions of doubly spin-labeled TRX in vitro. By directly comparing the distance distribution with classical MTSSL, it could be demonstrated that the genetically encoded spin-labeled amino acid is a valuable tool for the determination of the structure and dynamics of proteins in DEER experiments. The reduction kinetics of the spin-labeled amino acid was thoroughly assessed in bacterial cell culture and during cell lysis to establish guidelines for an optimized biosynthesis of spin-labeled proteins.
First hints pointing towards a serine protease-mediated control of reductive proteins could be gathered. Furthermore, a first functional test of the evolved PylRS/tRNAPyl pair could be shown in mammalian cells.
This developed method offers new perspectives for future in cell applications. In first experiments, spin-labeled proteins could be selectively detected in live E. coli cells.
However, reduction is the limiting factor, which leads to loss in signal intensity and thus leading to low modulation depths in DEER experiments (hampering in cell distance measurements). Further efforts should focus on enhancing the stability of new amino
67 acid analogues by chemical synthesis, and on the identification of cellular components responsible for the reduction of nitroxides. By increasing the steric hindrance of the nitroxide moiety with e.g. ethyl groups, the stability might potentially be increased.[213]
This could lead to higher integrity degrees of doubly-labeled proteins, thus increasing the data quality in DEER experiments. By altering the chemical structure to improve the in vivo stability, the integrity degree of doubly-labeled protein samples could potentially be increased, opening new perspectives for future in cell EPR DEER measurements.
Alternatively, a protective group strategy could be applied to the spin-labeled amino acid. This could render the nitroxide species inert during cellular protein expression, overcoming the current limitation of low integrity degrees caused by reductive processes.
By employing e.g. a photolabile protecting group strategy,[214] reduction of nitroxide spin labels could be prevented, making this approach compatible with time-consuming protein expression in mammalian cell culture. This way, photocaged, spin-labeled amino acids could be site-specifically incorporated into proteins and deprotected in an non-invasive manner, giving the experimentalist spatio-temporal control over nitroxide formation in live cells.
A complementary strategy could follow up on the identification of the responsible proteins orchestrating the reduction of nitroxides in bacterial cells. First experiments suggest an involvement of serine proteases in the degradation pathway of proteins causing the reduction of nitroxides in cells and lysates. Activity-based protein profiling could potentially further illuminate the cause of reduction by pull-down assays. This might give indications about possible targets for genomic knock-outs. A first step towards the identification of proteins involved in the reduction of nitroxides was made. 2D-SDS-PAGE analysis revealed the up-regulation of several proteins, during the biosynthesis of spin-labeled proteins. These hit candidates have to be further evaluated.
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