Understanding the structural and mechanistic details of protein-DNA interactions that lead to cellular defence against toxic metal ions in pathogenic bacteria can lead to new ways of combating their virulence....
Recently,
site-directed Cu2+ labeling has emerged as
an incisive biophysical tool to directly report on distance constraints
that pertain to the structure, conformational transitions, and dynamics
of proteins and nucleic acids. However, short phase memory times inherent
to the Cu2+ labels limit measurable distances to 4–5
nm. In this work we systematically examine different methods to dampen
electron–nuclear and electron–electron coupled interactions
to decrease rapid relaxation. We show that using Cu2+ spin
concentrations up to ca. 800 μM has an invariant effect on relaxation
and that increasing the cryoprotectant concentration reduces contributions
of solvent protons to relaxation. On the other hand, the deuteration
of protein and solvent dramatically increases the duration of the
dipolar modulated signal by over 6-fold to 32 μs. Based on this
increase in signal longevity, distances up to 9 nm and beyond can
potentially be measured with Cu2+ labels.
Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu(II)-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters...
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