Troels Schwarz-Linnet:
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This thesis is divided into two chapters. The first chapter concerns the continued work on detection and biophysical characterisation of post-translational modifications of an archaea esterase from Sulfolobus islandicus. The second chapter concerns development of in silico methods for rapid analysis and acquisition of NMR relaxation dispersion data.
Archaea esterase EstA from Sulfolobus islandicus
This work is a continuation from previous work in the group of Associate Professor Qunxin She at Danish Archaea Centre - University of Copenhagen.
The project is a comparative study of a thermophile-expressed and mesophile-expressed Sulfolobus islandicus esterase EstA. A considerable amount of effort has focused on eliminating some minor discrepancies in constructs and establish a purification protocol for E. coli expressed EstA, with equal solubility and dimer formation properties as the Sulfolobus islandicus expression. We also found it necessary to refine the previous described protocol for enzyme activity assays, since there exists a range of chemical challenges under elevated temperatures.
We did indeed find the thermophile-expressed EstA being a target for 1-3x lysine methylation post-translational modifications. We also confirm that the enzyme Archaeal Lysine Methyl Transferase (aKMT) is the responsible for this distribution of methylation of lysines. Via isoelectric focusing gels, we also established that Sulfolobus islandicus expressed proteins contains subpopulations of more acidic species. During the writing of this thesis, we found early literature describing these subpopulations as being a product of deamidation of asparagine residues. All mass spectromic data acquired under this project has been re-examined during writing of this thesis. Mass determination of previous unknown peptides from an endoproteinase AspN digestion of Sulfolobus islandicus EstA, can in some examples be assigned to peptides where an asparagine has been deamidated to aspartic acid. The in silico AspN digestion map of EstA, needs to allow cleavage of peptide bonds N-terminal to aspartic acid residues and asparagine to confirm these masses.
Our comparison between thermophile-expressed and mesophile-expressed EstA only shows minor differences. The differences between the EstA construct is most notable, when the enzyme has been exposed to stressconditions as elevated temperatures in detergent solution. These minor differences resemble results presented in other literature. An effort has been given to present and review literature hypotheses for the explanation of lysine methylations and compare the methods used in the different articles.
Rapid analysis and acquisition of NMR relaxation dispersion data
Performing a complete NMR relaxation dispersion analysis can be a challenging process. We engaged in a collaborative software development with other NMR departments in Europe, to further expand and optimise the NMR software relax. All mathematical operations in relax was carefully optimised to work in 5-dimensional data structures, which increased the calculation speed of relax by a factor 20-40x times.
We review how the software MddNMR efficiently can reconstruct sparsely recorded NMR relaxation dispersion data. We find that such datasets can be recorded at 1/4 of normal acquisition time without comprising the quality. The quality of the reconstruction is performed on both NMR peak intensity, as well as complete relaxation dispersion analysis. Only few articles within the field of non-uniform sampling expands the analysis further than peak intensities. This work can as such be used as a resource of comparison for the future development of methods in non-uniform sampling. The ability to reconstruct the NMR data depends on quality of the sample. We propose a method to assess the safe non-uniform sampling level, by sampling a fully recorded HSQC spectrum at different sparsity levels.
All data from the non-uniform sampling project was calculated in relax to ensure the highest quality of NMR dispersion analysis.