Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

Abstract 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 (K D) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm CuII K Ds 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.

With the increasing complexity and scope of the biomolecular structures being studied, pulse dipolar electron paramagnetic resonance (PDEPR) spectroscopy is an attractive technique that can complement crystallography,N MR spectroscopy,o rc ryo electron microscopy with nanometre distance constraints.S imilar to Fçrster resonance energy transfer (FRET), PDEPR does not require crystallisation, is not size-limited, and is performed in solution. It has been used to investigate the structure and dynamics of proteins and nucleic acids on alength scale of 1.5-16 nm [1] including multicomponent systems, [2] intermolecular domain interactions, [3] contribute distance constraints for structural modelling, [4] and mechanistic insights. [5] Furthermore,P DEPR can be used to monitor complexation [6] and so principally can couple structural information to binding equilibria. [7] Commonly,pairs of paramagnetic moieties,s uch as nitroxide radicals,a re sitespecifically conjugated with thiol side-chains of cysteines introduced at strategic positions via site-directed mutagenesis. Covalent attachment of the commercial methanethiosulfonate spin label (MTSL, Figure 1a)t oc ysteines results in the modified amino acid R1 bearing aspin-labelled side-chain. A major strength of this methodology is the opportunity to measure distances between identical labels.
Nonetheless,s pectroscopically orthogonal spin labels, such as transition metal ions, [8] lanthanides, [9] and triarylmethyl-based spin labels, [10] have gained increasing attention for use in conjunction with nitroxides.T his is appealing Figure 1. a) The structure of the modified cysteine residue R1 (top) and the Cu II -NTAs pin label, coordinated to the d-nitrogen atoms of the imidazoler ings of aprotein dH site (bottom). b) Double-dH (I6H/ N8H/K28H/Q32H GB1) construct in cartoon representation (PDB: 4WH4), [14a] with the Cu II -NTAs pin labels and coordinating dH sites in stick representation and Cu II ions as blue spheres. c) Raw RIDME trace (black) and background fit (red) for the measurement of 75 mm double-dH protein in the presence of 100 mm Cu II -NTAs pin label and d) distance distribution corresponding to the raw RIDME trace shown in (c).
because it can expand the accessible information content of as ingle multi-labelled sample. [11] Importantly,m ost labelling strategies rely on conjugation to cysteinet hiols, [12] which makes orthogonal site-specific labelling problematic.T o overcome this,g enetically encoded labels can be used, though as non-canonical amino-acids, [13] which can be more structurally perturbing than the post-translational modification of natural amino-acids,a nd can also restrict the yield of label incorporation.
Recently,Cu II -labelling using genetically encoded doublehistidine (dH) motifs, [14] introduced in a-helices (at residue positions i and i + 4), and b-sheets (at residue positions i and i + 2)h ave emerged as alternatives to nitroxides for pulse EPR applications. [15] Significantly,the chemical orthogonality of histidine coordination to covalent cysteine modification brings systems previously unamenable to standard labelling (for example,t hose containing essential disulfide bridges) into reach. Furthermore,b ipedal attachment and ar educed number of rotatable bonds between the paramagnetic centre and the protein backbone reduces the conformational flexibility of dH labels compared to R1, leading to dramatically improved precision in macromolecular distance measurements. [15] This makes dH-Cu II apowerful tool for the study of subtle conformational changes [16] that may otherwise be hidden in the intrinsically broad inter-label distributions observed with common spin labels.
However,asCu II -labelling of dH sites is non-covalent, the binding equilibrium is described by ad issociation constant (K D ). Ap oor dH affinity for Cu II would lead to compromise on two fronts:i )either the efficiency of protein labelling would be consistently low,orii) the excess of Cu II -label would be very large,leading to asignal dominated by free label. This would diminish the modulation depth (D)and make retrieval of the dipolar signal challenging as instrumental artefacts and background become more dominant. Another limitation is the reduced sensitivity of the routinely used pulse electronelectron double resonance (PELDOR) [17] experiment when applied to paramagnetic metal ions due to their large spectral widths.I nt his work, the 5-pulse relaxation-induced dipolar modulation enhancement (RIDME) experiment [18] is used to mitigate this sensitivity issue.B yr efocusing the dipolar interaction using astochastic spin flip rather than amicrowave pulse,t he common pulse excitation bandwidth limitation of PDEPR is overcome,l eading to significant sensitivity enhancement. In the case of RIDME measurements between nitroxides and metal ions,the detection of the nitroxide spin improves the signal while also allowing for the addition of excess Cu II ,t hereby improving occupation of the dH site without significantly reducing sensitivity,asfree Cu II will not contribute to the EPR echo signal. However,the addition of large excesses of Cu II will detriment sensitivity by shortening transverse relaxation and steepening the background. K D s have previously been estimated for Cu II -iminodiacetic acid (IDA) and Cu II -nitrilotriacetic acid (NTA) complexes binding to the protein dH sites used in this work. [15] Ag roup G Streptococcus protein G, domain B1 (GB1) tetra-histidine (double-dH) construct I6H/N8H/K28H/ Q32H [15a] is investigated by RIDME before the investigation of the individual dH sites through two dH/R1 constructs with dH sites in a b-sheet (I6H/N8H/K28R1) and an a-helix (I6R1/ K28H/Q32H), respectively.Lastly,weinfer asub-mm binding affinity from 5-pulse RIDME experiments at sub-mm protein concentrations.
The5 -pulse RIDME experiment (see the Supporting Information for details) on homo or hetero spin-pairs relies on detected spins (A spins) accumulating ap hase factor by longitudinal relaxation (with time constant T 1 )o fp artner spins (B spins) during the mixing-time interval T mix .W hen varying the position of T mix in the pulse sequence,t he phase factor manifests as am odulation of the detected refocused electron spin echo by the dipolar coupling, w AB ,w hich is proportional to the inter-spin distance r AB À3 . [19] Forasystem consisting of two electron spins S = 1 = 2 (such as Cu II or nitroxide), the T mix -a nd T 1 -dependent modulation depth (D Tmix )isg iven by: Foradouble-dH construct, only proteins with both dH sites occupied will contribute to the experimental modulation depth (D). With increasing Cu II concentration D Â D Tmix À1 will first increase while the dH sites are not saturated;t herefore, most detected Cu II will be dH-bound and contribute to D. However,once the dH sites saturate,all additional Cu II will be free in solution and reduce D towards 0a st he Cu II concentration tends to infinity (see the Supporting Information). This is overcome in the orthogonally labelled case,where quantitatively bound nitroxide is detected. Here, D Â D Tmix À1 reports the loading with Cu II ,a nd D will tend asymptotically to the theoretical limit of 0.5, as Cu II tends to infinite concentration, since the excess of Cu II does not contribute to the signal. This means that D Â D Tmix À1 as af unction of Cu II concentration is described by aq uadratic binding equation: where [P] t and [M] t are the concentrations of total protein and metal complex, respectively. RIDME measurements were performed on the double-dH GB1 construct (Figure 1b)at75mm protein concentration in presence of 100 mm Cu II -NTA ( Figure 1a). Thes ensitivity is estimated to be af actor of approximately 100 superior to PELDOR (see below). However,e stimating K D from Cu IIdetected PDEPR has several pitfalls.S peciation of Cu II ions into free and bound species leads to different transverse and longitudinal relaxation behaviour and EPR spectra for the different Cu II species.T his means the contributions to the signal will depend not only on the stoichiometric factors but also on the spectral position that is detected and the actual dipolar evolution time and experiment repetition rate.Inthis light, we refrained from quantifying K D sf rom Cu II -detected PDEPR. Nevertheless,i nf avourable cases deviations between species can be negligible or are readily determined from independent experiments.
In an approach to independently investigate the K D values of both dH sites via Cu II -nitroxide RIDME modulation depths,c onstructs I6R1/K28H/Q32H and I6H/N8H/K28R1 GB1 were designed (Figure 2a and b, respectively), produced and characterised (see the Supporting Information). In analogy to previous studies,t he binding strength can be estimated from PDEPR modulation depths by adding increasing amounts of titrant. [6a] Forp ractical reasons pseudo-titrations (as at itration with discrete samples prepared for each data point in the series) were performed. Initial predictions of concentration ranges consistently underestimated the Cu II loading and this led us to incrementally decrease protein and spin label concentrations.S peculation that the change in cryoprotectant and buffer conditions used here (50 mm phosphate + 50 %ethylene glycol in contrast to 50 mm N-ethyl morpholine + 20 %g lycerol used previously) [15] might cause the increased affinity were disproved (see the Supporting Information). Titrations at 25 and 75 mm protein concentrations led to K D estimates of below 3 mm for all four combinations of both spin labels (Cu II -IDAand Cu II -NTA) with both constructs.T he lowest estimate was below 500 nm for I6R1/K28H/Q32H and Cu II -NTA. It is important to note that at protein concentrations two orders of magnitude above the estimated K D ,o ur approach does not allow ap recise determination (see the Supporting Information).
Thus,a pproximate Cu II -loading was inferred from RIDME traces measured for both constructs at 5 mm protein and spinlabel concentration, confirming significant loading at low mm concentration with again the highest loading for I6R1/K28H/ Q32H and Cu II -NTA( see Supporting Information). Isothermal titration calorimetry (ITC) data predicted K D values in the range from 5t o4 2 mm for all construct/Cu II -label permutations (see Supporting Information). This is in line with previous literature reporting low mm K D for dH-Cu II . [20] Importantly,t he ITC analysis also yields ab inding enthalpy that allows predicting the dissociation constant as af unction of temperature according to vantH off.H ere,b inding is exothermic so at lower temperature tighter binding is expected. DH in kcal mol À1 are À9.68 (NTA-6H8H), À7.54 (NTA-28H32H), À3.37 (IDA-6H8H), and À5.75 (IDA-28H32H). Closer inspection reveals that extrapolating to 235 to 240 K, the RIDME-determined K D sagree remarkably well with the ITC data. As RIDME experiments are performed at 30 K, there will be no dissociation or association in the frozen sample,s ot he RIDME data will reflect the binding equilibrium when the dynamics of binding and releasing are frozen (see Supporting Information). These extrapolations,p revious binding studies, [6c] and the ITC data all suggest that the binding equilibrates fast. Even when snapfreezing aroom temperature sample by immersion into liquid nitrogen, the equilibrium reflects a5 0to6 0Klower temperature.
With the aim of unequivocally demonstrating the sub-mm K D of I6R1/K28H/Q32H and Cu II -NTAi nf rozen samples and the feasibility of RIDME studies at these concentrations ap seudo-titration was measured at 500 nm I6R1/K28H/ Q32H, in the presence of 100 to 8100 nm Cu II -NTAs pin label (Figure 2c). Qualitatively, D increases with increasing Cu II -NTA, before plateauing towards 0.5, as is expected according to Equation (1). Thef it of the experimental D Â D Tmix À1 values to Equation (2) approximates a K D value of 150-300 nm (see the Supporting Information), which is consistent with the prediction at higher protein concentration. Interestingly,t he observed D values do not reach the asymptotic limit of 0.5;r ather, values tend to approximately 0.45. [6a] Thus,w ec hose to scale D and to employ bivariate fitting to Equation (2) for the binding isotherm ( Figure 2d). Scaled results are virtually identical to fixing the asymptotic value of Equation (1) to 0.45 (see the Supporting Information).
In summary,i th as been demonstrated that due to the exothermic nature of the binding process,t he a-helical i and i + 4 dH site has as ub-mm affinity for Cu II -NTAu nder cryogenic EPR conditions.T his has important implications for the use of Cu II -chelators as spin labels,and particularly for their application to double-dH constructs.T he increased sensitivity afforded by both Q-band RIDME in Cu II -nitroxide and Cu II -Cu II systems compared with Cu II -Cu II PELDOR experiments is truly promising;o ur sensitivity determination [21] gives improvements by factors of approximately 150 and approximately 100, respectively (see the Supporting Information). Similarly,R itsch et al. have shown that Cu II -nitroxide RIDME outperforms PELDOR by asignificant margin, even in as tate-of-the-art ultra-wide band- . The histidine residues that form the dH site and the R1 labels are shown as sticks;t he Cu II -NTA label is modelled with the Cu II -centre shown as ab lue sphere. c) Background-corrected 5-pulse RIDME traces of 500 nm I6R1/K28H/ Q32H GB1 measured as apseudo-titration with 100-8100 nm Cu II -NTA. The experimental data are coloured accordingtothe plot legend with the corresponding fits shown as dashed black lines. d) Experimental D as aquotient of D Tmix [calculated using Eq. (1)],extracted from the Cu II -NTAp seudo-titration series, (Figure 2c), as af unction of Cu II -NTAconcentration (red dots) with the fitted binding isotherm in blue. width spectrometer. [22] Importantly,RIDME is also less prone to orientation selection than PELDOR, facilitating distance extraction. [23] This sensitivity range opens up the possibility of routine distance measurements at greatly reduced protein concentration, making new systems accessible and potentiating new science. [24] This work further demonstrates how RIDME can be used to measure binding equilibria, and subsequently determine K D sr emotely via the dipolar interaction under experimental EPR conditions.T his strategy is important as it couples structural and thermodynamic information for protein-ligand interactions and could be applied to ligand-gated systems.T he approach is complementary to calorimetric methods,s uch as ITC,e specially in the study of high-affinity interactions,w here other methods of structural investigation are often sensitivity-limited. Used together, we anticipate that dH sites and the RIDME experiment can expand the PDEPR tool-box to the study of protein systems previously beyond reach. In conclusion, dH affinity for Cu IIchelators is not limiting for PDEPR studies,a nd K D values can be directly estimated from 5-pulse RIDME data and agree well with low-temperature extrapolated ITC data. Furthermore,this study demonstrates that using spectroscopically orthogonal spin labels,such as Cu II -NTAand nitroxide, in combination with sub-mm PDEPR experiments is feasible.