Supporting Information

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1 Supporting Information Wiley-VC Weinheim, Germany

2 ryllium Displacement of + from Strong ydrogen Bonds T. Mark McCleskey *, Timothy S. Keizer, Deborah S. Ehler, Dilip N. Asthagiri, Lawrence R. Pratt, Brian L. Scott*. Los Alamos National Laboratory. MS J514 Los Alamos, NM In loaded transferrin preparation and stoichiometry measurements. Indium-loaded transferrin. To a solution of apotf in 0.1M EPES/5 mm sodium bicarbonate buffer, p 7, was added 3 equivalents of In(N 3 ) 3. A 10,000 NMWL Amicon Centricon TM YM-10 unit was used to remove unbound indium; the rententate was washed twice with buffer and then concentrated in the same Centricon unit. ryllium (8 equivalents) was added as a 1.1 M S 4 solution. A p of 7 was maintained. Stoichiometry experiment. ApoTf or holotf was dissolved in a buffer containing 25mM Tris and 85 mm NaCl, p 7. ryllium (20 equivalents) was added as a 1.5M S 4 solution. After 3 hr mixing, the solutions were filtered using Fisherbrand TM 0.45 um nylon syringe filters. The <0.45 um filtrate was then centrifuged in a 10,000 NMWL Amicon Centriplus TM YM-10 unit. The >10K rententate was washed twice with buffer and then taken through the entire filtration sequence again. The resulting >10K rententate was then analyzed for Tf and beryllium concentrations. Theoretical calculations and simulations. We calculated the energetics of the reaction with Gaussian 03. For the hydration calculations, we assumed a dielectric continuum description and used a boundary element method. To keep the calculation tractable, we adopted the following style of assigning basis functions. In the minimization, frequency and thermal-vibrational correction calculation steps we used the following basis sets: carbons and hydrogens bonded to carbons, the 6-31G basis; hydrogens bonded to noncarbon atoms get the 6-31G(p) basis; oxygens, nitrogens, and beryllium gets 6-31+G(d,p) basis. For the single point energy calculations and for calculating partial charges via the ChelpG procedure, the following basis sets were used: carbons and hydrogens bonded to carbons get 6-311G basis; hydrogens bonded to non-carbon atoms get the 6-311G(p) basis; oxygens, nitrogens, and beryllium gets G(2d,p) basis. Previously used radii are used for the atoms in the continuum dielectric calculations. The numerical results yield values of E, G corr, and u * of , , and 62.2 kcal/mol for SA - and , , and 60.1 kcal/mol for SA()( 2 ). For the reaction in scheme 1 of the manuscript the change in electronic energy ΔE = 0 kcal/mole; this is consistent with the fact that the salicylate anion has to first lose a proton, but recovers that unipositive charge via binding to + species. ΔGcorr = 1.7 kcal/mole, suggesting that thermal vibrational effects favor importantly,_μ * = 1.4 kcal/mole. ydration effects play a crucial role that cannot be ignored. The Gibbs free energy for the reaction listed in Scheme I of the manuscript was calculated using the following thermodynamic cycle:

3 2 2 2 (g) + 3 C C (g) 2 (g) (g) 2 3 C C ΔG 1 ΔG 2 ΔG3 ΔG 4 ΔG g (aq) + 3 C C (aq) ΔG aq 3 C C 2 2 (g) (g) ΔG aq = ΔG g + ΔG 3 - ΔG 2 - ΔG 1 + ΔG 4 = kcal/mol The individual Gibbs free energies were calculated using Gaussian03 at the DFT/6-31G(d) level. The molecules denoted with (g) were optimized and had their energies calculated in the gas phase. The molecules denoted (aq) were optimized and had their energies calculated in water using the PCM model. Salicylic acid is an excellent model system since it has a rigid backbone structure, a strong hydrogen bond (NMR shift of 16.3 ppm) and a high pka (>13). The crystallographic --- angle has been reported as º and resides in between the ideal 180 o for a linear hydrogen bond and the 120 o that would exist in a perfect six member ring. This represents an ideal situation for beryllium to approach with its tetrahedral geometry that can bind both oxygens in a geometry that completes the six member ring. Scheme II shows a general view of a [( 2 ) 3 ()] + complex approaching a SA - and displacing it with water as a product. We have calculated the net change in free energy for the overall reaction to be 3.1 kcal/mole. The picture in Figure 1 of the manuscript was generated using the WebLab Viewer Pro 4.0 software, copyrighted by Molecular Simulations Incorporated of San Diego, California. The starting point was the b-chain of the 1PB5 crystal structure downloaded from the RCSB Protein Databank ( The beryllium dication and water molecules were placed in the structure at the strong hydrogen bond sites (Table

4 2) using the draw option. The clean option was used to optimize the first coordination geometry of all beryllium sites. NMR experiments. All NMR spectra were recorded on a Bruker Avance 500 Mz spectrometer using tunable broadband probe with z-gradients. All spectra were processed using Bruker XWINNMR 2.6 software. Initial sample contained 0.36 mm solution of transferrin in 0.1M EPES, 5mM sodium bicarbonate buffer, p 7.0, in 90%2/10%D2, and was titrated with 1.11M solution of 2+ solution to achieve final 2+ concentrations of 0.9, 1.8, 2.7, 3.6, 4.5 and 5.4 mm, corresponding to equivalents of 2+ /protein molecule. 1 spectra were obtained using WATERGATE pulse sequence, with 14ppm sweep width and transmitter centered at water resonance. 512 scans were recorded with 16k points/spectrum. The recycle delay was set to 3s. The data were zero-filled to 32k points and apodized with 1z exponential function before Fourier transform. Spectra were indirectly referenced to DSS (0.00 ppm) through the frequency of water resonance (4.70 ppm) Figure 1S. 9 NMR of apotransferrin with 5,10 and 15 equivalents of. 9 spectra were obtained using simple one pulse sequence with 10ppm sweep width centered at 3 ppm scans were recorded for each experiment with 2048 points/spectrum. The recycle delay was set to 3 s, for total acquisition time of hrs/experiment. The data were zero-filled to 8k points and apodized with 4z exponential

5 function before Fourier transform. All spectra were indirectly referenced to the resonance of 2+ (aq) at 0.0 ppm. Figure 2S. 9 NMR of In loaded transferrin with 5,10 and 15 equivalents of. Figure 3S. Comparison of the 9 NMR of In and apotransferrin with10 equivalents of. The 9 NMR shifts for the small molecule mimics corresponding to hydrogen bonds in Table 1 of the manuscript are listed in the Table 1S below. The molecules listed in Table 1S were all optimized using Gaussian03 at the F/3-21G(d) level. The optimizations and energy calculations were performed in the gas phase. Also listed are 9 NMR shifts for model systems used to validate the calculations, which agree quite

6 well with the experimental values for several strong hydrogen bonded, oxygen donor systems.. The 9 nmr line shifts were calculated using the GIA method. Gaussian03 is referenced in the paper. The mimic Aq 2 (Ac) 2, which represents 2 out of 12 sites in apotf, has a slightly higher predicted shift of 2.33 ppm. There is no peak at 2.33 ppm in the NMR, but two sites are probably not sufficient to observe a signal. Complex 9 (experimental), ppm 9 (calculated), ppm Aq 2 (Phe)(Ac) Aq 2 (Me)(Ac) Transferrin: (broad) 1.66, 1.72 Aq 2 (Ac) (IPA) 2 ( 2 ) (DBA)( 2 ) , , 2.54 (SSA)( 2 ) IPA= 2-hydroxy-isophthalic acid, DBA= 2,3- dihydroxy benzoic acid, SSA= 5-sulfosalicyclic acid. Table 1S. Calculated and measured 9 NMR shifts [F/3-21G(d); GIA method; Gaussian 03 5 ] Sites with an -- distance less than two times the sum of the Van der Waals radii (3.2 Å) were considered.