Lanmodulin peptides – unravelling the binding of the EF-Hand loop sequences stripped from the structural corset

Lanmodulin (LanM), a naturally lanthanide (Ln)-binding protein with a remarkable selectivity for Lns over Ca(ii) and affinities in the picomolar range, is an attractive target to address challenges in Ln separation. Why LanM has such a high selectivity is currently not entirely understood; both specific amino acid sequences of the EF-Hand loops and cooperativity effects have been suggested. Here, we removed the effect of cooperativity and synthesised all four 12-amino acid EF-Hand loop peptides, and investigated their affinity for two Lns (Eu(iii) and Tb(iii)), the actinide Cm(iii) and Ca(ii). Using isothermal titration calorimetry and time-resolved laser fluorescence spectroscopy (TRLFS) combined with parallel factor analysis, we show that the four short peptides behave very similarly, having affinities in the micromolar range for Eu(iii) and Tb(iii). Ca(ii) was shown not to bind to the peptides, which was verified with circular dichroism spectroscopy. This technique also revealed an increase in structural organisation upon Eu(iii) addition, which was supported by molecular dynamics simulations. Lastly, we put Eu(iii) and Cm(iii) in direct competition using TRLFS. Remarkably, a slightly higher affinity for Cm(iii) was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring and therefore a reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.


Solvents and Chemical Reagents
Chemicals used in this work were obtained from the following commercial sources: Merck KGaA, Sigma-Aldrich, abcr, Thermo Fisher Scientific, Acros Organics, VWR or TCI. Lanthanide salts were purchased at a purity of at least 99.9%. Amino acids for SPPS (solid phase peptide synthesis) were purchased as Fmoc-protected compounds with standard residual protecting groups from ORPEGEN Peptide Chemicals GmbH and Sigma-Aldrich. Resins for SPPS were purchased from Rapp-Polymere. The Pierce™ quantitative fluorometric peptide assay kit (23290) was purchased from Thermo Scientific. In general, pro analysi (p.A.) quality solvents were used. For preparative HPLC purification HPLC grade solvents and for analytical HPLC/HPLC-ESI-HRMS analysis LC-MS grade solvents were used. For all experiments ultrapure water (type 1, pH 5.6, 18.2 MΩ/cm at 25 °C) was used. Therefore, demineralised water was further purified using a Synergy® UV system from Merck Millipore®. All chemicals were used as supplied, unless stated otherwise, and handled according to potential hazards and toxicity. For information on the hazard potential of peptide coupling reagents see literature. 1

Solid Phase Peptide Synthesis (SPPS)
Solid phase peptide synthesis was performed using a CEM Liberty Blue™ automated microwave-assisted peptide synthesiser under standard Fmoc conditions.
Peptides (see Table S1) were synthesised on a 0.1 mmol scale using commercially purchased Fmoc-Glu(tBu)-Wang resin (capacity: 0.30 mmol/g) in order to receive C-terminal carboxylic acids. The pre-loaded resins were swollen in DMF (4 mL) for at least 1 h prior to use. Standard Fmoc-amino acids were prepared as 0.

Concentration
The assay was carried out similarly to the protocol 3 provided online by Thermo Scientific in black 96-well plates at room temperature using a Biotek H1MF hybrid multi-mode reader. The excitation wavelength was set to 390 nm, the emission wavelength to 475 nm, the gain to 72 and the reading height was 5.35 mm. The provided peptide digest assay standard (1 mg/mL) was diluted in water to give a 12-point standard-curve (see Figure S2). The synthesised peptides were dissolved in water (2 mg/mL gross weight) and diluted to 300 μg/mL gross weight. The standardcurve as well as all samples were performed in triplicates. To 10 μL sample/standard, 70 μL of the provided buffer and 20 μL fluorometric reagent were added, followed by 1 min orbital shake and 5 min incubation at room temperature. The obtained data were analysed with Gen 5™ 3.10. For fitting the standard curve, a degree two polynominal function was used (y = a + bx + cx 2 ). The obtained standard curve and parameters are shown in Figure  S2 and were used to determine the mean net peptide concentration in µg/mL; the calculated values are shown in Table S3. Figure S2 Standard curve of the quantitative fluorometric peptide assay with polynomial fit to determine the mean net peptide concentration.

Determination of the Target Peptide Concentration
The mean net peptide concentration of the triplicates in μg/mL was used together with the HPLC purity (see Table  S2) and the molecular weight (see Table S1) of the target peptide to determine the target peptide concentration in μmol/L by using Equation 1. The obtained target peptide concentrations are shown in

Binding Studies
Caution: When working with actinides special precautions must be taken. All samples containing actinides were handled in a radioactive controlled area following the official regulations.

Metal stock solutions
CaCl2 · 2 H2O solutions were prepared as 100 mM and 1 M stock solutions in water and LnCl3 · n H2O (Ln = Tb, Eu) solutions as 10 mM in water; all stock solutions were stored at −20 °C until usage.
The Cm(III) ( 248 Cm (97.3%)/ 246 Cm (2.6%)/ 245 Cm (0.04%)/ 247 Cm (0.02%)/ 244 Cm (0.009%)) stock solution was obtained by converting a 7.3 mM stock solution of long-lived Cm isotopes in 1.0 M HClO4 into a 7 mM Cm(III) solution in 1 M HCl (concentration determined by liquid scintillation counting) by adding 1.5 mL 5 M NaOH to 500 μL of the perchlorate solution until a clearly alkaline pH was reached. The solution was centrifuged (10 min, 13000 rpm) and the supernatant discarded. The solid residue was dissolved in 500 μL 1 M HCl and the described process repeated four times.

CD Spectroscopy (CD)
Circular dichroism (CD) spectra were recorded in a Hellma Analytics® quartz cell (110-QS, 1 mm pathlength) on a JASCO J-810 CD spectropolarimeter in MOPSO buffer (10 mM, 100 mM KCl, pH 6.6) at room temperature between 190 nm and 250 nm. The scanning speed was 50 nm/min, the data integration time 4 s, the data pitch 0.5 nm, the bandwidth 1 nm and the number of accumulations 3. A baseline spectrum of the used buffer was recorded and subtracted from the obtained spectra. Metal titration experiments were performed by adding the respective metal chloride (CaCl2· 2 H2O or EuCl3 · 6 H2O) in increasing amounts (14 steps, at least a 5-fold excess was reached) to a solution of the respective peptide. To avoid dilution effects, the peptide (EF1: 74.2 µM, EF2: 73.1 µM, EF3: 81.8 µM, EF4: 77.0 µM) and buffer concentration (10 mM MOPSO/ 100 mM KCl, pH 6.6) was kept constant during the experiment by using a metal solution that contained the same buffer and peptide concentration as the analyte solution. The given peptide concentration was corrected with the obtained ITC factor (see Section 4. 3) The measured ellipticity in mdeg was converted to molar ellipticity (θ) using Equation 2 were d is the pathlength in cm and M the used peptide concentration in mol/L.

Isothermal titration calorimetry (ITC)
ITC titration experiments were performed at 25 °C on a Malvern Panalytical MicroCal PEAQ-ITC instrument. The reference cell was filled with water, the stirring speed set to 750 rpm and the initial delay to 60 s. The cell was either loaded with 280 µL of a 100 µM or of a 20 µM peptide solution (concentration based on the results of the HPLC analysis and the fluorescence assay) in 10 mM MOPSO/ 100 mM KCl buffer (pH 6.6). For Eu(III) titration experiments in total either 38 injections (first and 20 th injection 0.4 µL; residual injections 2 µL) of a 1.5 mM or 39 injections (first injection 0.4 µL; residual injections 1 µL) of a 600 µM EuCl3 solution in the same buffer system in time intervals of 150 s were performed. Ca(II) titration experiments were performed using the 39-injection set-up with a peptide concentration of 20 µM in the cell and 3 mM Ca(II) solution in the syringe. Before loading the cell/syringe they were both equilibrated with the used buffer. Analogously performed titrations using the same settings of EuCl3 or CaCl2 in buffer to buffer were used as background measurements; ITC data is always plotted as the differential power (DP) versus time. The obtained data was analysed with MATLAB ® . To optimize data analysis, raw ITC data were pre-treated using the following workflow to reduce noise: Raw ITC files were read into MATLAB ® (Fig. S3 A). The full thermogram was cut into individual injections and those were arranged parallel to each other to form a matrix (Fig. S3 B). The offset of each injection was roughly estimated by its initial value to get rid of significant variance caused by this offset. Such a matrix can be used with singular value decomposition (SVD) as provided by MATLAB ® . To account for changes in peak shapes within the injection series, the number of factors for rebuilding the data was set to higher values as the expected number of chemical species, e.g. 4 eigenvalues and eigenvectors. 4,5 The residues and the reconstructed matrix are shown in Fig. S3 C and D. Afterwards the rows of the matrix were back-transformed to the thermogram and individual injections were integrated. Corresponding plots with and without noise reductions are compared in Fig S3 E and F. Our ITC data are a superposition of 3 different Eu(III)-peptide complexes. At the very beginning of the titration the reproducible small increase (see Fig S4 A-D) is indicative for 2:1 peptide-Eu(III) complex. With increasing Eu(III) concentrations, the first dominant complex should be the 1:1, whereas at high Eu(III) concentration even two metals can bind to one peptide forming a 1:2 peptide-Eu(III) complex. The 2:1 peptide-Eu(III) complex is not dominant at all and only responsible for the small increase at the beginning. It is not present in significant amounts in our system (see lower speciation plot in Fig S4 A-D) and therefore its fitted thermodynamic parameters strongly depend on the initial values. The fitted enthalpies are −2e-6 (A), −25 (B), −40 (C) and −80 kJ/mol (D). Therefore, we decided to exclude (dark squares) not only the first point of each titration, but also the second and fit only two complexes (the 1:1 and 1:2 peptide-Eu(III) complex). The obtained thermodynamic parameters for the 1:1 and 1:2 peptide to Eu(III) complex are shown in Table S4. The obtained value for N (given in Table S4) was used as a correction factor for the earlier determined peptide concentrations (see Section 3.3).

Figure S3
Noise reduction procedure for ITC data. Individual injection peaks of the raw data (A) were rearranged in matrix form (B). This matrix was decomposed using SVD. With this procedure the noise (C) is separated from the purified signal (D). The differences of integrated curves with and without this noise reduction procedure is shown in (E) and (F), respectively.

Figure S4
The integrated heats of all Eu(III) to EF-hand titrations show similar features. The overall curves were dominated by the existence of an 1:1 EF/Eu(III) complex. Additionally, all curves show a significant offset from zero after subtraction of the measured background. This is indicative for the existence of an 1:2 EF/Eu(III) complex. Furthermore, at the very beginning of the titration experiment, an initial increase in ΔH is observed. In this part of the titration, the peptide is in excess to Eu(III). Thus a 2:1 EF/Eu(III) complex can be formed, in which two peptides coordinate one metal. Unfortunately, the thermodynamic parameters of this complex cannot be reliably fitted (figures above exemplary for EF1/Eu(III)). Depending on the starting conditions, the ΔH/mol of this complex varies from −2e-6 (A) over−25 (B) and −40 (C) to −80 kJ/mol (D). As can be seen from the minor fractions of this complex in the underlying speciation, this complex is essentially negligible and only responsible for the slight increase. Therefore, we decided to exclude the second injection from the analysis and focus on the thermodynamic characterization of the 1:1 and 1:2 EF/Eu(III) complex as shown in E (left: ITC fits and right: speciation).

Time-resolved Laser-induced Fluorescence Spectroscopy (TRLFS)
All titration experiments were performed in MOPSO buffer (10 mM, 100 mM KCl, pH 6.6 for Eu(III) and Tb(III) experiments, pH 6.0 for Cm(III) experiments) at 25 °C in stirred (1200 rpm) 2 mL or 4 mL 10 mm pathlength Hellma Analytics quartz cells equipped with a screw cap and septum. The cuvette was placed in a cuvette holder which was connected via a light guide to a spectrograph (Andor, SR-303i-A). For recording the spectra an ICCD (Andor iStar, DH320T-18U-63) was used. For Eu(III), Tb(III) and Cm(III), the excitation wavelengths (Ekspla, NT230, ~5 ns pulse) were set to 394 nm (grating: 300 l/mm), 220 nm (grating: 300 l/mm) and 396 nm (grating: 600 l/mm), respectively. Competition experiments with Cm(III) and Eu(III) were performed with an excitation wavelength of 393.9 nm (grating: 300 l/mm For clarity all peptide concentrations in the text above are given without taking the correction factor, determined by ITC, into account. However, the correction factor (see Table S4) was applied during the analysis of all data sets. All data sets were individually analysed in MATLAB ® . The raw data were loaded using the sif-reader 6 Detailed data analysis was performed with PARAFAC (N-way toolbox for MATLAB ®7 ) with modification described elsewhere 8,9 . To match a speciation an adopted version of a MATLAB ® script from Smith 10 was used.
Control experiment for Tb(III) TRLFS: To test the stability of the peptides during the experimental set-up when using a laser irradiation of 220 nm a 10 µM solution of EF2 in water was prepared and split in two parts of which one was irradiated for 1 h. The samples were compared by mass spectrometry using the following set-up: HPLC-ESI-HRMS measurements were performed on an Agilent HPLC 1260 Infinity II system (G7129A 1260 vialsampler, G7111B 1260 Quat Pump, G7717C 1260 DAD HS, G7116N 1260 MCT) coupled to a UHD Accurate Mass Q-TOF LC MS system (G6538A). All measurements were performed at 25 °C as direct measurements or by using an Agilent Zorbax C18 column (250 mm×4.6 mm, 5 µm) with a flow rate of 0.4 mL/min. As an eluent system a mixture of water/acetonitrile with 0.1% formic acid was used. All data was obtained in positive mode. No evidence for the decomposition of the peptide was found. However, the concentration observed in the MS seemed to decrease after 1 h of irradiation, which is not understood yet. Furthermore, the high consistency between the different data sets using Eu(III), Cm(III) and Tb(III) and reproducibility within the Tb(III) experiments strongly suggest that the peptides do stay intact while being irradiated at 220 nm.

Molecular dynamics (MD) simulations
2.1 Methodology MD simulations and data analyses were performed using AMBER 15 program package 11 with ff99SB force field applied on the protein. For Eu, 12-6-4 Lennard-Jones-type parameters developed by Metz et al. were employed. 12 Two different starting structures were used (one from NMR solution structure 13 (PDB 6mi5), the other without any initial restraints). The protonation state of the peptides was adjusted to model physiological pH. Note, that Na(I) ions were added for the system without Eu(III) to make the system electrostatically neutral. Then, TIP3P waters were added with a minimum water layer thickness of 10 Å. 500 steps of steepest descent and 500 steps of conjugate gradient -with 500 kcal mol −1 Å −1 harmonic restraint on the peptides -were initially conducted, after which 1000 steps of steepest descent and 1500 steps of conjugate gradient were performed without constraints. 40 ps of heating of the system from 0 to 300 K with 10 kcal mol −1 Å −1 harmonic restraint on the peptides, after which another 1 ns preconditioning run was performed at 300 K without restraint on the solutes. Finally, a 200 to 350 ns MD simulation run was performed in a periodic boundary condition in an NPT ensemble from which the last 100 ns trajectory was taken. Simulations were terminated and restarted every 5 ns. The SHAKE algorithm, a 2 fs time integration step, 12 Å cut off for non-bonded interactions, and the particle mesh Ewald (PME) method were used. MD trajectory was recorded at every 50 ps. Oxygen atoms within 2.7 Å distance from metal centres were defined as coordinated atoms.      Figure S15 PARAFAC decomposition of TRLFS data for peptide (0-22.7 µM) to Tb(III) (0.25 µM) titration. From left to right deconvoluted spectra (normalised to the 5 D4→ 7 F6 transition), lifetimes of the observed species, speciation and t0 spectra (blue to red from first to last titration step) normalised to the 5 D4→ 7 F6 transition; λex (Tb)= 220 nm. Colour code in all graphs: Green = Tb(III)aquo ion and pink = 1:1 peptide to Tb(III) complex.