Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase.

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.

Site-directed attachment of photoexcitable spin labels for light-induced pulsed dipolar spectroscopy.

Photoexcited triplet states are gaining popularity as spin labels in pulsed electron paramagnetic resonance (EPR) spectroscopy. Here, we demonstrate that the fluorophores Eosin Y, Rose Bengal and Atto Thio12 are suitable markers for distance determination by laser-induced magnetic dipole (LaserIMD) spectroscopy in proteins that lack an intrinsic photoexcitable center.

Optimising broadband pulses for DEER depends on concentration and distance range of interest.

EPR distance determination in the nanometre region has become an important tool for studying the structure and interaction of macromolecules. Arbitrary waveform generators (AWGs), which have recently become commercially available for EPR spectrometers, have the potential to increase the sensitivity of the most common technique, double electron-electron resonance (DEER, also called PELDOR), as they allow the generation of broadband pulses. There are several families of broadband pulses, which are different in general pulse shape and the parameters that define them. Here, we compare the most common broadband pulses. When broadband pulses lead to a larger modulation depth, they also increase the background decay of the DEER trace. Depending on the dipolar evolution time, this can significantly increase the noise level towards the end of the form factor and limit the potential increase in the modulation-to-noise ratio (MNR). We found asymmetric hyperbolic secant (HS{1,6}) pulses to perform best for short DEER traces, leading to a MNR improvement of up to 86 % compared to rectangular pulses. For longer traces we found symmetric hyperbolic secant (HS{1,1}) pulses to perform best; however, the increase compared to rectangular pulses goes down to 43 %.