Monitoring Complex Formation by Relaxation‐Induced Pulse Electron Paramagnetic Resonance Distance Measurements

Abstract Biomolecular complexes are often multimers fueling the demand for methods that allow unraveling their composition and geometric arrangement. Pulse electron paramagnetic resonance (EPR) spectroscopy is increasingly applied for retrieving geometric information on the nanometer scale. The emerging RIDME (relaxation‐induced dipolar modulation enhancement) technique offers improved sensitivity in distance experiments involving metal centers (e.g. on metalloproteins or proteins labelled with metal ions). Here, a mixture of a spin labelled ligand with increasing amounts of paramagnetic CuII ions allowed accurate quantification of ligand‐metal binding in the model complex formed. The distance measurement was highly accurate and critical aspects for identifying multimerization could be identified. The potential to quantify binding in addition to the high‐precision distance measurement will further increase the scope of EPR applications.

Biomolecular complexes are often multimersf ueling the demandf or methods that allow unraveling their composition and geometrica rrangement. Pulse electron paramagnetic resonance (EPR) spectroscopy is increasingly applied for retrieving geometrici nformationo nt he nanometer scale. The emerging RIDME (relaxation-induced dipolar modulation enhancement) technique offersi mproved sensitivity in distance experiments involving metal centers (e.g. on metalloproteins or proteins labelled with metal ions). Here, am ixture of as pin labelled ligand with increasing amountso fp aramagnetic Cu II ions allowed accurate quantification of ligand-metal binding in the model complex formed. The distance measurement was highly accurate and critical aspects for identifying multimerization could be identified. The potentialt oq uantify binding in addition to the high-precision distance measurement will further increasethe scope of EPR applications.
The ever-growing complexity of structures underpinning functional materials and the molecular basis of our understanding of health and diseasef uels an increasing demand for new (bio)physical tools elucidating the composition and geometry of large assemblies or complexes. In recent years, pulse EPR has proven of utmost value fors tudying complex biological systemsa nd revealing topology information not accessible by other methods. Biological targets of pulsed electron-electron double resonance (PELDOR or DEER) [1] spectroscopy involve cutting-edge applications in probing conformational changes during protein translocation [2] and mechanosensation [3] as well as identifying the role of non-coding RNAs in protein sequestration,s torage and release. [4] The impact of pulse EPR on structuralr esearch has sparked ar enaissance of EPR methodology involving new hardware, [5] pulse sequences [6] and computationalt ools. [7] Relaxation-induced dipolar modulation enhancement (RIDME) [8] is particularly useful when measuring distances to paramagnetic metal centers [9] and the introduction of ad ead-time free sequence [6c] combined with rigorous experimentalb enchmarking have led to am ultiplication of applications. [6c, 10] These embrace chemical model systems [8, 9b, 11] as well as model proteins. [6c, 10a, c] In both RIDME and PELDOR, as et of spins (A) is detected while an inversion of as econd set of spins (B) selectively introduces the dipolar spin-spin interaction between Aa nd Bs pins (w dd ). Varying the timing (t)o ft he B spin inversion causest he As pin signal to oscillate with the interaction frequency (cosw dd t,F igure 1, panel A, left) that encodes the distance between the spins parameter-free. In RIDME the excitation of Bs pins is based on stochastic spin relaxation (longitudinalr elaxation, T 1 )r ather than caused by am icrowave pulse as in PELDOR. The RIDME 'mixing time' (T mix )d efines the time interval that permits for stochastic Bs pin relaxation. For very broad spectra that metal ions often display the fraction of inverted Bs pins (l)c an be much larger in ar elaxation driven RIDME experiment hence boosting sensitivity. [10b, 12] The excitation of forbidden electron-nuclears pin transitions can result in ESEEM (electron spin echo envelope modulation) and obscure the desired dipolar modulation. This is especially   KGaA. This is an openaccessarticleunder the termsoft he Creative Commons AttributionL icense, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. relevant in deuterateds ystems. However,d euteration allows substantially extending the spin-spin distance range and suppressing the unwanted background signald ecay. [12] Several methods for ESEEM suppression and removal have been reported. [10a, b, 12] Keeping these challenges under control, RIDME is a very appealing alternative to the establishedP ELDOR method. [1] In PELDOR the number of electron spins per nano-object can be retrieved from the depth of the dipolar oscillations (D, Figure 1, panel A, left). [13] Assessing multimerization degrees in as imilar fashion via relaxation-based pulse EPR would be av aluable addition to the distance information in the data. Here, we seek to experimentally test whether RIDME allows probing the degree of ligand binding to aparamagnetic metal ion template. Scenarios where the quantification of binding might be adding significant insight include the uptake of ap aramagnetic cofactor by as pin-labelledp rotein allowing at the same time to measure the distance as well as loading with paramagnetic metal ions. Especially when diamagnetici ons are substituted this might be useful as binding constant might differ. Similarly,R IDME in electron transfer systemsm ight quantify the amount of charge transfer as only the oxidized (or reduced) speciesm ight be paramagnetic. The quantification of multiple binding sites could indeedb eh elpful for quantifying metal loading (e.g. for metal-metal distance measurements in both biological systemsand inorganic supramolecules).
As am odel bindinge quilibrium between an itroxide (NO) spin carrying ligand and ap aramagnetic metal ion bearing template, aN O-labelled 2,2':6',2''-terpyridine ligand (L) [13a] and paramagnetic Cu II ions (electron spin S = 1 = 2 )w ere employed. The Cu II /L ratio was varied systematically from 0.0 to 1.0 in steps of 0.1 while keeping the absolutel igand concentration constant ( Figure 1, panel A, right). [13a] The hypothesis to be tested assumes:1 )L not bound to Cu II (Figure 1, panels Aa nd B L)w ill not experience RIDME and justd isplay ab ackground signal, while 2) L bound to Cu II (Figure 1, panelsAa nd B [CuLX n ] 2 + and [CuL 2 ] 2 + ,w ith X n representing ns olvent molecules filling the Cu II coordination sphere) will show RIDME.
If the signal is the linear superposition of contributions from bound and unbound L,t he depth of the dipolar modulations D will reportt he fraction of ligand bound to am etal ion. The Cu II /L ratio 0.0 will correspond to pure background signal while from 0.1 to 0.5, 20 %t o1 00 %o fL will be boundi nt he dimer species[ CuL 2 ] 2 + with any residual L being free in solution. Depending on the cooperativity of binding, [13a] addition of further metal leads to either the coexistence of [CuL 2 ] 2 + and solvated Cu II or their comproportionation to [CuLX n ] 2 + .I n either case, for Cu II /L ratios 0.5 to 1.0, D should stay constant as all L will be boundt oafast-relaxing metal center.I ti si mportant to note that as econd L binding to the Cu II ion after the first is not expected to alter the Cu II -NO RIDME modulation depth.B ased on the crystal structure of L [13a] we expect the Cu II -NO distance distribution to peak at 2.6 nm. [11a] Measurements at Q-band frequencies ( % 34 GHz) in deuterated matrixt om aximize sensitivity showed substantial ESEEM and made it essential to minimize these unwanted contributions. [10a, b] Suppression by increasing the pulse lengths proved unsuitable for quantification of modulation depths( see Supporting Information, SI). ESEEM removal by deconvolution (deliberatelyf orfeitingt he dipolar modulation in ar eference experiment still containing ESEEM and subsequentd ivision;s ee SI for details) were tested using as econd experiment reducing the temperature from 30 Kt o1 5K (Figure 2, left and SI) or reducing T mix from 200 mst o5ms (Figure 2, right and SI).
Both deconvolution methods yieldedv isually ESEEM-free RIDME data and Tikhonovr egularization [7b] resultedi nd istance distributionss howings harp peaks at the expected 2.6 nm distance. For the temperature-basedm ethodn od ipolar modulation couldb er ecovered forr atio 0.1. Importantly,n either methods howedt he expected trend in the modulationd epths (a linear increase to ap lateau from 0.5 on, Figure 2, bottom, blue triangles), but ac ontinuous increase of the Cu II -NO modulation depth from 0.0 up to 1.0 Cu II /L ratios was observed. Fitting the experimental modulation depths to expected trend with D as free parameter leads to root mean square deviation (rmsd) to the fit of 25 %o fD for temperaturea nd 16 %o fD for T mix deconvolution (Figure 2, blue triangles and SI). This large deviation is attributed to the coexistence of [CuL 2 ] 2 + and [CuLX n ] 2 + for ratios between 0.5 and 1.0 (Figure 1, panel A,  right).B oth speciesh aving different longitudinal relaxation times (SI) the deconvolution experiment will have the dipolar couplings uppressed differentlyb etween samples and this will lead to partial removalo fm odulation depth by the division. The experimental trend in modulation depth can be simulated as af unctiono fT 1 and T mix (Figure 2, grey crosses and SI).
As this hampers the robustness of deconvolutionm ethods for quantitative modulationd epths withoutapriorik nowledge of components and their relaxation times, experimentsa voiding deconvolution were tested. Owing to the high 1 HL armor frequency ( % 52 MHz at 1.2T )E SEEM is not visible in protonated samples and pronouncedR IDME oscillations are observed (data in SI). Here, the RIDME modulation depth encodes the ratio of the radical boundt ot he paramagnetic metal ion. For ratios 0.0t o0 .5 the Cu II -NO modulation depth increases with the fraction of metal-bound ligand and for ratios 0.5 to 0.9 D stays virtually constant,a sa ll ligands are tethered to metal ions. Ratio 1.0 showing ano utlier that could be due to the specific sample or experiment. Nevertheless, there is good agreement with the model (rmsd between data and fit 8% of D). The expected 2.6 nm distance was found (see SI).
While the quantification of RIDME modulation depths becomes feasible, protonated samples severelyl imit the achievable maximum distance and resolution. Fortunately,d uring the investigationr eported here, Yulikov and co-workersp ublished the successful averaging of unwantedE SEEM modulations [12] abolishing the need for deconvolutiona nd allowing to use deuterated samples leadingt oc learly superior results (Figure 3 and SI). Notably,a na pparent modulation depth is already retrieved from artifacts present in as ample without any added metal. Thus, small modulation depthss hould be interpreted with caution.
The RIDMEm odulation depth increases from ratios 0.0 to 0.5 while after that ratio andu pt o1 .0 D was largely constant (rmsd to modelled modulation depths7%o fD). The expected 2.6 nm Cu II -NO distance was found also here.
The results were confirmed by performing X-band measurements in protonated and deuterated matrix averaging 1 Ha nd 2 HE SEEM respectively (data in SI). Although 2 HE SEEM modulations couldn ot be completely diminished by averaging at 9GHz, the measurements could follow the percentageo f ligand bound to Cu II ions (SI).
This work demonstrates that the RIDME modulation depth does encode the number of radicals tethered to fast relaxing paramagnetic centers with spin 1 = 2 andt hus allows quantification. Even in as ystemw ith weaker binding affinity D will re-flect the fraction of ligand with bound metal;h owever, this will not necessarily be all metal added. As the free metal only contributes to background the modulation depth quantifies bound metal and thus, the binding constant when the total metal ion concentrationi sk nown.Q uantifyingc omplex formation from modulation depthsi ns ystems with varying relaxation times via deconvolution methods hasb een found to be unsatisfactory.H ere, the choice of mixingt ime andt emperature will alter the weights of contributions of the individual species. Avoiding deconvolution methods and using sufficiently long mixing times, quantification becomes practical as demonstrated here. The suppression of ESEEM by averaging [12] has been instrumental in resolving distances and measuring relative percentage of the spin pair from as ingle RIDMEe xperiment. As deuteration is ap rerequisite for reliably resolving long distances and the complications caused by variationsi n relaxation times of the paramagnetic metals complicate deconvolutionm ethods, reliable interpretationo fD values from Qband RIDME should be pursued in deuteratedm atrix using the nuclear modulation averaging approachw ith as ufficiently long mixing time.