Infrared studies : Method development and binding of ligands to pyruvate kinase

University dissertation from Stockholm : Department of Biochemistry and Biophysics, Stockholm University

Abstract: Infrared spectroscopy is a valuable technique for the study of ligand induce change in biomolecules. Our development of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. We use this method to understand the binding of phosphoenolpyruvate (PEP) and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. To investigate the effect of the protein environment on the bound PEP, we used labeled PEP, which helped assign and evaluate the infrared absorption bands. The effects of different divalent and monovalent ions on PEP binding to PK were also studied. We could demonstrate that the β-sheets were perturbed differently with Na+ as compared to the other monovalent ions. The pattern of structural changes does not correlate with the activity profiles of the monovalent ions. Thus, it seems unlikely that the ion effects on activity are due to the ion effects on the structure of the PEP:PK complex. Comparing different divalent ions, a particularly large conformational change and a more homogeneous binding mode was observed with Mn2+ and attributed to a more closed conformation of the complex. The allosteric effect of fructose 1, 6 bisphosphate (FBP) on PEP binding to PK in presence of various ions (Mg2+, Mn2+, K+, Na+) was studied. The experiments indicated that the conformational change of PEP binding to PK:Mg2+:K+ in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy of the closed state of PK. The affinity for PEP increased in presence of Mg2+ and K+. No allosteric effects were observed with the other ion combinations Mn2+/K+ and Mg2+/Na+. A method of ligand binding by observing a change in water absorption was developed and established with four different proteins. The results suggest that the decrease of water absorption is due to the release of bound water into the bulk during the ligand binding process, which can be a used as label-free indicator of ligand-protein binding.

  This dissertation MIGHT be available in PDF-format. Check this page to see if it is available for download.