Biophysical characterization of tryptophan mutants in carbonic anhydrase from Neisseria Gonorrhoeae

• Main Author: Dunbring, Daniel
• Format: Others
• Language: English
• Published: Linköpings universitet, Institutionen för fysik, kemi och biologi 2007
• Subjects: Carbonic Anhydrase; Neisseria Gonorrhoeae; Tryptophan Mutation; purification; Fluorescence; CO2 hydration; Biochemistry; Biokemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-9797

In this project the aim has been to study the model protein carbonic anhydrase in Neisseria gonorrhoeae, a bacterium whose carbonic anhydrase has great similarities both structurally and functionally with the human form. By measuring and comparing the wild type of NGCA with mutants lacking one of the four tryptophan residues it can be seen what effect these tryptophans has on stability and activity and then compare with the known data of HCA II to learn more about their differences and similarities. The results from the stability and activity measurements are that the wild type is by far the most stable protein with W141L mutant coming thereafter. From Trp-fluorescence and CO2-hydration measurement a clear two-transition steps (N→ I→ U) can be seen. This differs from earlier data where it instead only was a one-transition step for the wild type (N→U). The data is also very reliable and gives in most cases a perfect fit to the line. We also see this two-transition step for the other mutants stable enough, strengthening the theory further. One fact that could be drawn from all the measurements is that when an intermediate is formed the ability for the enzyme NGCA to perform it’s catalytically ability is disabled. Another thing is that the purification scheme of HCA II is not optimal to be directly applied to NGCA, despite the similarity in secondary and tertiary structure.