Tritium NMR studies provide a convenient way of obtaining detailed information about conformational equilibria, dynamic processes and specific interactions in protein-ligand complexes provided that suitably 3H-labelled molecules are available. In this study [7,9-3H]- and [3',5',7-3H]folic acid, and [3',5',7-3H]methotrexate were synthesised and the NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analysed as a function of pH (DHFR-folate complexes) and temperature (DHFR-methotrexate complexes). From these data it was possible to obtain further evidence about the orientation of the pteridine ring in the complexes, and to monitor the dynamic processes in the bound ligands. In the 3H NMR spectra of the ternary complexes of the 3H-labelled folic acids with DHFR and NADP+, each labelled tritium gave rise to multiple signals, confirming previous findings that there are three interconverting, pH dependent, conformational forms of bound folate (forms I, IIa and IIb) in the ternary complex. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the methotrexate binary complex and also the ternary complex with NADPH were shown to exist as a single conformational state with the benzoyl ring flipping rate being too slow to give a single averaged signal for the 3',5'-tritium nuclei over the temperature range 283 - 313 K. 3h{1h} Nuclear Overhauser enhancement experiments have been conducted on the small molecules, [3H]dimethyl sulphoxide, [3',5',7-3H]folic acid and [3',5',7-3H]methotrexate as a prelude to 3H-1H heteronuclear NOE experiments on binary and ternary complexes formed using Lactobacillus casei DHFR and the ligands [3',5',7-3H]methotrexate and [3',5',7-3H]folic acid.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:240033 |
| Date | January 1994 |
| Creators | Curtis, Nicola |
| Publisher | University of Surrey |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://epubs.surrey.ac.uk/842983/ |
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