Abstract. 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 %.
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.
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