Conformational dynamics and function of proteins studied by hydrogen/deuterium exchange mass spectrometry

Any attempt to understand the function of proteins requires an investigation of the conformational dynamics and their relation to activity. H/D exchange experiments coupled with proteolytic peptide mapping and mass spectrometric detection are among the most powerful tools for monitoring the structure and dynamics of proteins.;Another aspect of this thesis is the investigation on the role of conformational dynamics on phosphorylation-induced activation of VraR, a key process against antibiotics in Staphylococcus aureus. VraR is highly dynamic, the phosphorylated form (VraR-P) shows much less extensive deuteration. The changes in deuteration within the receiver domain are consistent with a Y-T coupling mechanism. In analogy to NarL, the activation of VraR is thought to involve separation and subsequent reorientation of the two domains, thereby exposing the alpha8-turn-alpha9 element so that it can engage in DNA binding. The current work suggests that this switch is triggered by a reduction in the effective length of the linker through enhanced hydrogen bonding. In addition, separation of the two domains may be favoured by the establishment of intra-domain contacts at the expense of previously existing inter-domain bonds. alpha9 appears to be packed against the receiver domain in non-activated VraR. Support is presented for alpha1 as a dimerization interface in VraR-P, whereas protein-protein interaction for the non-phosphorylated protein is impeded by extensive disorder in this region.;Overall, this thesis illustrates now MS-based H/D exchange measurements can provide detailed insights into the function of proteins, yielding information that is complementary to data obtained from X-ray crystallography or NMR spectroscopy.;Keywords: Conformational dynamics; enzyme kinetics; hydrogen/deuterium exchange; electrospray ionization; mass spectrometry; pepsin digestion; peptide mapping; enzyme; catalysis; phosphorylation-induced activation; resistance to antibiotics; two-component system; signal transduction; carboxypeptidase B; thermolysin; lysozyme; vancomycin-resistance associated regulator (VraR); VraR-P; methicillin-resistant or "multi-drug resistant" S. aureus (MRSA); vancomycin-resistant S. aureus (VRSA).;It remains an open question in how far the conformational dynamics of enzymes are different during catalysis and in the substrate-free state. Comparative measurements carried out on carboxypeptidase B and thermolysin in the absence and in the presence of their corresponding substrates result in H/D exchange kinetics that are indistinguishable. This observation is consistent with the view that catalytically relevant motions pre-exist in the "resting state". It thus appears that enzyme function is mediated by intrinsic dynamics. The backbone fluctuation of free thermolysin indicates that elements composed of active site are rigid to moderately flexible, which may reflect a carefully tuned balance that is required for function.