WeO-08


PROBING THE 3D STRUCTURE OF PROTEINS WITH MASS SPECTROMETRY

Carol V. Robinson

Oxford Centre for Molecular Sciences, South Parks Road, Oxford, OX1 3QT


The development of mass spectrometry to study proteins from solutions in which protein remains in its native state during introduction to the mass spectrometer has had major impact on many protein structural studies. Using the combination of hydrogen exchange labeling and mass spectrometry (HX-MS) protein folding pathways of proteins have been studied and provided important information concerning events on protein folding pathways1. HX-MS has also been used for studying non-covalent protein-ligand interactions and has provided additional information relating to conformational changes which occur in proteins upon ligand binding2. Furthermore, the conformation of protein ligands bound to molecular chaperones has been determined by HX-MS3.

Recent studies have shown that HX-MS also has a role to play in the study of amyloid forming proteins. Using wild type and variant transthyretin we have shown changes in the stability of the non-covalent tetramer which have been linked to its tendency to form amyloid fibrils. The effect of ligand binding to the tetramer on the stability of the complex has also been investigated by MS. In addition the stability of two variants of human lysozyme which form amyloid in vivo have been studied by HX-MS and clearly show that the variants unfold under physiological conditions to expose hydrophobic core. This transient unfolding of the amyloidogenic proteins, together with information from other biophysical techniques, suggests a mechanism by which these protein molecules can associate to form amyloid fibrils.

References
1. "Cooperative Elements in Protein Folding Monitored by Electrospray Ionization Mass Spectrometry" S.D. Hooke, S.J. Eyles, A. Miranker, S. Radford, C.V. Robinson and C.M. Dobson, J. Am. Chem. Soc. 117, 7548-7549, (1995).
2. "Probing the Nature of Non-covalent Interactions by Mass Spectrometry", C.V. Robinson, E. Chung, B. Kragelund, R. Aplin, F. Poulsen and C.M. Dobson, J. Amer. Chem. Soc. 118: 8646-8653, (1996).
3. "Conformation of GroEL-bound a-lactalbumin probed by Mass Spectrometry" C.V. Robinson, M. Grob, S.J. Eyles, J.J. Ewbank, M. Mayhew, F.U. Hartl, C.M. Dobson and S.E. Radford Nature 372, 645, (1994).