A Bioorganometallic Approach to Study Histidine Kinase Autophosphorylations

Abstract

Auto-phosphorylation of bacterial histidine kinases PhoR, PhoQ, and EnvZ has been investigated using adenosine-5'-[γ-ferrocene] triphosphate (Fc-ATP) as a cosubstrate for the first time. The study has been carried out in solution and on surface. Results from biochemical multiplex assay and surface electrochemical/optical methods are consistent, which successfully demonstrates that Fc-ATP is an efficient cosubstrate for histidine kinase auto-phosphorylations. The study also has discovered that the concentration of Fc-ATP influences the autophosphorylation efficiency. This developed methodology will provide a powerful tool in studying such biological processes towards further understanding of the involved mechanism.

Phosphorus-Based Probes as Molecular Tools for Proteome Studies: Recent Advances in Probe Development and Applications

“…This may be caused by the binding of Fe 2+ or the ferrocenyl moiety to the sensory domain of the kinase. The interference of divalent cations (Mg 2+ ) in autophosphorylation has been previously observed . Additionally, the presence of human serum albumin leads to a current density decrease (50–70%), but the signal remains sufficiently strong for quantification …”

Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications

“…They explained this observation by the fact that at certain concentrations of Fe 2+ cations or ferrocenyl groups, these moieties may have an inhibitory effect on the proteins by interacting with their sensory domain. 145 Despite these advances, radioactive ATP derivatives remain the most widely implemented method for the study of HK and RR activity. 144,146,147 To globally map the Mycobacterium tuberculosis ATP-binding proteome and identify potential ATP-related targets for the development of novel antibacterial drugs, Wolfe et al used desthiobiotin-ATP, a lysine reactive acyl ATP, in a chemoproteomic approach (Fig.…”

Mechanistic Studies of Bioorthogonal ATP Analogues for Assessment of Histidine Kinase Autophosphorylation

“…29,30 Kraatz and coworkers have utilized a 5′-γ-ferrocenyl-adenosine triphosphate, another aminophosphate analogue, for monitoring histidine kinase activity electrochemically. 31 Here, we sought to establish an understanding of the molecular requirements for the design of efficient HK probes based on the ATP scaffold. We investigated three ATP derivatives, both experimentally and computationally, that possess γ-phosphates conjugated to a terminal propargyl group, γ-propargyl-ATP (Probe O), γ-[(propargyl)-thio]-ATP (Probe S) and γ-[(propargyl)-imido]-ATP (Probe N; Figure 2).…”

“…We anticipated that the decreased electronegativity of N in comparison to O could provide similar advantages as the S atom used in our previous probe design. Indeed, Pflum and coworkers have described the use of ATP-based probes containing a terminal aminophosphate derivative for the study of mammalian kinases. , Kraatz and coworkers have utilized a 5′-γ-ferrocenyl-adenosine triphosphate, another aminophosphate analogue, for monitoring histidine kinase activity electrochemically …”