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MSc Analysis of selectively 15N labelled Ppib, proinsulin connecting peptide and DnaG-C by NMR

Domenii publicaţii > Chimie + Tipuri publicaţii > Tezã de masterat (nepublicatã)

Autori: Bogdan Bancia

Editorial: 2004.

Rezumat:

The three-dimensional structure determination of proteins represents an important step towards understanding their biological function and thus their roles in living organisms. Using a combination of multidimensional NMR techniques three different biomolecules were analyzed in the present study, E. coli peptidyl – prolyl cis-trans isomerase PpiB, proinsulin connecting peptide and DnaG-C.

15N-HSQC spectra were recorded of PpiB which had been expressed without further purification in a cell-free expression system with amino acid selective isotope labelling. Comparison of spectra before and after ultrafiltration indicated that labelled metabolic by-products are of low molecular weight. Therefore, the labelled protein signals are easily distinguished from those of metabolites.

The structure analysis of the proinsulin connecting peptide included the assignment of 1H, 13C and 15N NMR resonances using 2D NMR, measurement of T1 (1H) relaxation times and comparison of chemical shifts with sequence dependent chemical shift prediction. The results showed that the peptide assumes a random-coil conformation in aqueous solution.

The NMR resonance assignment of the C-terminal domain of E. coli DnaG primase was performed with the goal to assess its structural flexibility in solution. A 2D TOCSY experiment recorded with long mixing time identified mobile residues at the N- and C-terminal ends. A 15N-HSQC experiment and a number of triple resonance 3D experiments (HNCA, HNCO, CACB(CO)HN, HNCACB, HNCAHA and NOESY-15N-HSQC) were used for the backbone assignment. {1H}15N NOEs were measured to characterize the mobility of the protein backbone.

Overall, these projects demonstrate the power of NMR spectroscopy for the study of biomolecules in solution, in particular with respect to the analysis of structural flexibility which presents an important aspect of their biological function.

Cuvinte cheie: protein structure determination