Paramagnetic Nuclear Magnetic Resonance (NMR) Studies on the Calcium Binding Site of [Alpha]-Lactalbumin

Paramagnetic NMR has drawn interest as a technique for extending the range of systems that can be investigated. The purpose of this study was to determine whether a combination of paramagnetic two-dimensional and relaxation techniques allows for assigning resonances corresponding to residues forming the metal binding site of proteins in which the metal is coordinated exclusively by amino acid side chains and which have been previously inaccessible to assignment. α-Lactalbumin (LA) is a calcium binding protein with relatively broad NMR signals; this broadness has prevented complete assignment. LA has been the subject of many studies, because it can form a molten globule state which is closely related to one of the intermediates in protein folding. The assignment of LA\u27 s resonances could therefore serve as basis for experiments investigating protein folding. In a first approach, calcium was exchanged for gadolinium, which enhances relaxation. However, the properties of gadolinium turned out to be too strong to allow for discrimination between resonances. In a second approach, calcium was exchanged for ytterbium, which has strong hyperfine-shifting properties. The melting point of ytterbium LA was determined by observing the change in NMR peak intensities with temperature. Hyperfine-shifted resonances were assessed as completely as possible by one-dimensional NMR experiments, connected into spin-systems by two-dimensional NMR experiments, and their spin-lattice and spin-spin relaxation times were determined. Assignment of the residues constituting the metal binding site was then sought with the help of LA\u27s crystal structure and exchange experiments. Although the combined experimental data suggests one model of resonance assignments, they must be seen as tentative for three reasons. First, the crystal and solution structures of the metal binding site might differ significantly. Second, the relaxation enhancing properties of ytterbium might be strong enough to broaden the resonances of the closest protons to such an extent that they become invisible in the NMR spectrum. Third, different mechanisms of the protons closest to the metal binding site and those further away might govern relaxation which would complicate systematic interpretation of the data