Nickel and gtp modulate helicobacter pylori ureg structural flexibility

UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions

Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism

“Inhibition and aerobic inactivation kinetics of Desulfovibrio fructosovorans NiFe hydrogenase studied by Protein Film voltammetry”

International audienc

The quantitative applications of EPR spectroscopy in the study of structure-fonction relationships in metalloproteins

The quantitative study of EPR spectra given by metalloproteins raises a number of difficulties, especially in the case of multicenter systems in which the different signals generally overlap. In this paper are presented different methods that have been recently developed in the laboratory to study the relationship between structure and fonction in these systems

Single-crystal electron paramagnetic resonance study of cytochrome c3 from Desulfovibrio desulfuricans Norway Strain. Assignment of the heme midpoint redox potentials.

International audienceA single crystal of cytochrome c3 from Desulfovibrio desulfuricans Norway is studied by electron paramagnetic resonance at low temperature. The orientation of the principal axis corresponding to the largest g value is determined for the 12 heme groups in the crystal unit cell. The comparison of these directions to the normals to the heme planes, determined from the crystallographic data at 2.5 A resolution, gives strong evidence for the following assignment of the midpoint redox potentials to the heme groups H1 to H4, defined in the three-dimensional structure: -150 mV is assigned to H3, -300 mV to H4, -330 mV to H1 and -355 mV to H2. This assignment is in agreement with a partial correspondence previously established from an independent study performed on cytochrome c3 in solution.A single crystal of cytochrome c3 from Desulfovibrio desulfuricans Norway is studied by electron paramagnetic resonance at low temperature. The orientation of the principal axis corresponding to the largest g value is determined for the 12 heme groups in the crystal unit cell. The comparison of these directions to the normals to the heme planes, determined from the crystallographic data at 2.5 A resolution, gives strong evidence for the following assignment of the midpoint redox potentials to the heme groups H1 to H4, defined in the three-dimensional structure: -150 mV is assigned to H3, -300 mV to H4, -330 mV to H1 and -355 mV to H2. This assignment is in agreement with a partial correspondence previously established from an independent study performed on cytochrome c3 in solution

“Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase.''

International audienc

“In Rhodobacter sphaeroides respiratory nitrate reductase, the kinetics of substrate binding favors intramolecular electron transfer”

International audienc

“Access to the Active Site of Periplasmic Nitrate Reductase: Insights from Site-Directed Mutagenesis and Zinc Inhibition Studies”

International audienc