Studies of site-selective glycosylation of dihydrofolate reductase

Tey, Lai-Hock 2008. Studies of site-selective glycosylation of dihydrofolate reductase. PhD Thesis, Cardiff University.

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Abstract

Glycosylation is important for many molecular processes, although how glycosylation contributes to glycoprotein structure and function is not entirely clear. Other than that, the study of many of these events is complicated by the fact that natural glycoproteins normally occur as mixtures of glycoforms, therefore the isolation or synthesis of homogenous glycoproteins is an important task. Previous studies on deglycosylated proteins have shown that glycosylation reduces their catalytic activity and increase thermal stability. Therefore, we hypothesize that similar effects may be observed in the naturally unglycosylated dihydrofolate reductase from Escherichia coli (EcDHFR). Four different surface-exposed sites for the incorporation of a single cysteine residue were selected based on the protein crystal structure, which may or may not affect its dynamics. Homogeneous glycoproteins have been synthesized via chemoselective ligation of a glycosyl haloacetammide with the thiol of a cysteine, to produce site- selectively glycosylated forms of EcDHFR. Techniques such as mass spectrometry, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, ultraviolet (UV) visible spectroscopy and stopped-flow spectrophotometry have been used to identify and study the physical properties of different glycoforms of DHFR. Although there were some changes of the kinetic activity of the mutants of EcDHFR. the values were comparable to those of the wild-type protein. Interestingly, in four of the five cases studied. EcDHFRDM D87C, there was an increase in thermal stability upon site-selective glycosylation. The other mutants showed no effect. With the exception of the effect seen for the thermal stability of the D87C mutant, this is not in accordance with the original hypothesis. This suggest that the effect seen in the D87C mutant may be due to specific interactions of the carbohydrate moiety at certain points on the protein. An increase in resistance to thermal denaturation observed for proteins in sugar solutions may therefore also be due to binding of the sugars to specific sites on the protein. In conclusion, an effective method for the synthesis of homogeneous glycosylated and non-glycosylated proteins has been developed and applied to the site selective glycosylation of EcDHFR. The results also suggested that the kinetic properties of EcDHFR are not significantly affected by glycosylation. It may be the large effects in terms of protein stability which due to glycosylation only occur in naturally glycosylated proteins, and not in the naturally unglycosylated EcDHFR.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
ISBN:	9781303185519
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:33
URI:	https://orca.cardiff.ac.uk/id/eprint/55798

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