Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent CuII coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (KD) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm CuII KDs under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled CuII–nitroxide systems using a commercial Q‐band EPR instrument.
The study of ever more complex biomolecular assemblies implicated in human health and disease is facilitated by a suite of complementary biophysical methods. Pulse Dipolar electron paramagnetic resonance Spectroscopy (PDS) is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards PDS at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS using a combination of nitroxide and Cu
II
based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate Cu
II
-Cu
II
and nitroxide-nitroxide PDS measurements at protein concentrations below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDS measurements at short to intermediate distances (~1.5 - 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.
Protein interaction studies often require very low concentrations and highly sensitive biophysical methods. Here, we demonstrate that pulse dipolar electron paramagnetic resonance spectroscopy allows measuring dissociation constants in the nanomolar...
Pulse-dipolar EPR
is an appealing strategy for structural characterization
of complex systems in solution that complements other biophysical
techniques. Significantly, the emergence of genetically encoded self-assembling
spin labels exploiting exogenously introduced double-histidine motifs
in conjunction with Cu
II
-chelates offers high precision
distance determination in systems nonpermissive to thiol-directed
spin labeling. However, the noncovalency of this interaction exposes
potential vulnerabilities to competition from adventitious divalent
metal ions, and pH sensitivity. Herein, a combination of room-temperature
isothermal titration calorimetry (ITC) and cryogenic relaxation-induced
dipolar modulation enhancement (RIDME) measurements are applied to
the model protein
Streptococcus sp.
group G. protein
G, B1 domain (GB1). Results demonstrate double-histidine motif spin
labeling using Cu
II
-nitrilotriacetic acid (Cu
II
–NTA) is robust against the competitor ligand Zn
II
–NTA at >1000-fold molar excess, and high nM binding affinity
is surprisingly retained under acidic and basic conditions even though
room temperature affinity shows a stronger pH dependence. This indicates
the strategy is well-suited for diverse biological applications, with
the requirement of other metal ion cofactors or slightly acidic pH
not necessarily being prohibitive.
Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints.C ombining double-histidine motifs with Cu II spin labels can further increase the precision of distance measurements.Itisalso useful for proteins containing essential cysteines that can interfere with thiol-specific labelling.However,the non-covalent Cu II coordination approachis vulnerable to lowb inding-affinity.H erein, dissociation constants (K D )a re investigated directly from the modulation depths of relaxation-induced dipolar modulation enhancement (RIDME) EPR experiments.This reveals low-to sub-mm Cu II K D sunder EPR distance measurement conditions at cryogenic temperatures.W eshowthe feasibility of exploiting the doublehistidine motif for EPR applications even at sub-mm protein concentrations in orthogonally labelled Cu II -nitroxide systems using acommercial Q-band EPR instrument.
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