Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

We investigated pathogenic mutations relevant in dihydrolipoamide dehydrogenase (LADH; gene: Dld) deficiency, a severe human disease, to elucidate how they alter reactive oxygen species (ROS) generation and associated biophysical characteristics of LADH. Twelve known disease-causing mutants of human LADH have been expressed and purified to homogeneity from E. coli. Detailed biophysical and biochemical characterization of the mutants has been performed applying circular dichroism (CD) spectroscopy, nano-spray mass spectrometry (MS), calibrated gel filtration and flavin adenine dinucleotide-content analysis. Functional analyses revealed that four of the pathogenic mutations significantly stimulated the ROS-generating activity of LADH and also increased its sensitivity to an acidic shift in pH. LADH activity was reduced by variable extents in the mutants exhibiting excessive ROS generation. It is remarkable that in the P453L mutant, enzyme activity was nearly completely lost with a ROS-forming activity becoming dominant, whereas the G194C mutation, common among Ashkenazi Jews, resulted in no alteration in LADH activity but a gain in the ROS-generating activity. There have been neither major conformational alterations nor monomerization of the functional homodimer of LADH associated with the higher ROS-generating capacity as measured by CD spectroscopy and size-exclusion chromatography combined with nano-spray MS, respectively. The excessive ROS generation of selected LADH mutants could be an important factor in the pathology and clinical presentation of human LADH deficiency and raises the possibility of an antioxidant therapy in the treatment of this condition.