Kinetic analysis of copper transfer from a chaperone to its target protein mediated by complex formation

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Experimental section 2 Table S1. 1 H, 15 N chemical shifts of isolated apo-CopAab and in the presence of 2 equivalents of Cu(I)CopZ.4
Processing and analysis of MS experimental data was carried out using Compass DataAnalysis version 4.1 (Bruker Daltonik, Bremen, Germany).Samples for non-denaturing MS were prepared by first adding DTT (15 mM, Formedium) and removing excess reductant by passage down a G25 Sephadex column (PD10, GE Healthcare) in an anaerobic glovebox (Faircrest Engineering, O 2 concentration <2 ppm) using 20 mM ammonium acetate, pH 7.4 (Sigma) as the elution buffer.The protein sample was diluted with 20 mM ammonium acetate to a working sample concentration of 15 µM.To prepare Cu(I)-bound protein samples, a deoxygenated solution of Cu(I)Cl prepared in 100 mM HCl and 1 M NaCl was added to anaerobic, reduced CopZ or CopAab using a microsyringe (Hamilton) in an anaerobic glovebox.Unbound Cu(I) was removed by passage of the sample down a G25 Sephadex column (PD10, GE Healthcare) equilibrated with 20 mM ammonium acetate pH 7.4.Mass spectra were acquired using a Bruker micrOTOF-QIII electrospray ionisation (ESI) time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Coventry, UK), in positive ion mode and calibrated as above.Native protein samples were introduced to the ESI source via a syringe pump (Cole-Parmer) at 5 µL/min, and data acquired for 3 min, with ion scans between 50 -3000 m/z.MS acquisition was controlled using Bruker oTOF Control software, with parameters as follows: dry gas flow 5 L/min, nebuliser gas pressure 0.8 Bar, dry gas temperature 180 °C, quadrupole RF set at 2000 Vpp (25%) and 3200 Vpp (75%).Processing and analysis of MS experimental data was carried out using Compass DataAnalysis version 4.1 (Bruker Daltonik, Bremen, Germany).Neutral mass spectra were generated using the ESI Compass version 1.3 Maximum Entropy deconvolution routine over a mass range of 7200 -35000 Da.Exact masses are reported from peak centroids representing the isotope average neutral mass.Predicted masses are given as the isotope average of the neutral protein or protein complex, in which Cu(I)-binding is expected to be charge compensated. 2NMR Titration of apo-CopAab with Cu(I)-CopZ.NMR spectra were collected at 298 K on Avance 900 Bruker spectrometers operating at proton nominal frequencies of 900.13 MHz.Spectra were processed using the standard Bruker software (XWINNMR), and analyzed through the CARA software. 6Titrations of 15 N-labelled apo-CopAab with Cu(I)-CopZ in the presence of 1 mM DTT were performed by monitoring the 1 H-15 N HSQC spectral changes upon the addition of increasing amounts of the titrant.Aliquots were added in a Coy chamber under a nitrogen atmosphere at 25 °C.

Supplementary Tables
Table S1. 1 H, 15 N chemical shifts of isolated apo-CopAab and in the presence of 2 equivalents of Cu(I)CopZ.

Figure S2. LC-ESI-MS analysis of protein containing fractions from thermodynamic Cu(I)-transfer experiment.
Deconvoluted spectra corresponding to the first and second elution peaks from the Ni 2+ -affinity column as described in Figure 1 of the main paper.The first peak represents protein that did not bind to the column and consists almost entirely of CopAab (observed at 15911 Da, predicted mass 15911 Da).Some (His) 6 -CopZ (observed at 8403 Da, predicted mass 8402 Da) is present, but because CopZ ionises somewhat more efficiently than CopAab, the relative intensity of the two protein peaks represents an overestimation of the amount of CopZ present in this fraction.The second peak represents protein that eluted from the column only in the presence of imidazole (500 mM).Only (His) 6 -CopZ was observed, the vast majority as a monomer, with a small amount of disulfide bonded dimer (16802 Da).Only small changes in absorbance were observed.At longer time periods, scattering due to protein precipitation occured and so Cu(I)-transfer could not be followed further, but it is clear that little or no transfer of copper occurred during the experiment.If it is assumed that the loss of A 265 nm intensity of the Cu(I)-CopZ sample was due to Cu(I) transfer, extrapolation of the decay as a first order process gave an estimate of the dissociation rate constant of ∼ 6 × 10 -5 s -1 .This represents an upper limit because the decrease in Cu(I)-CopZ A 265 nm is very likely due to a combination of some Cu(I) dissociation and loss of protein due to, for example, adsorption onto the dialysis cassette membrane.

Figure S12 .
Figure S12.Species present in the mass spectra after copper transfer between CopZ and CopAab.
Figure S1.UV-visible absorption analysis of Cu(I)-binding to (His)6-CopZ.A) UV-visible absorption anaerobic titration of (His) 6 -CopZ (25 µM) in 100 mM Mops, 100 mM NaCl, pH 7.5.CopZ was pre-reduced with 15 mM DTT and passed down a G25 Sephadex desalting column (PD-10) in an anaerobic glove box to remove the DTT.Cu(I) was added as a 1 mM Cu(I)Cl solution, as previously described (1-3).B) Plot of ΔA 265 nm as a function of Cu(I)/CopZ.Solid and dotted lines respectively indicate distinct binding phases and the levels of Cu(I) at which they intersect.Like wild type CopZ, Cu(I) binding to (His) 6 -CopZ occurred in distinct phases: 0 -0.5, 0.5 -1.0, 1.0 -1.5, and > 1.5 Cu(I) ions per protein.The behaviour is essentially identical to that previously reported for the untagged protein (1), demonstrating that the Cu(I)-binding properties of CopZ are not affected by the presence of the Cterminal tag.

Figure S3 .
Figure S3.Measurements of Cu(I) dissociation from CopZ.CopZ, prepared at 0.5 Cu/protein, and apo-His 6 CopZ were placed into separate dialysis cassettes and both submerged in the same buffer solution.Here, the two proteins are unable to interact, and so transfer of Cu(I) from Cu-CopZ to apo-CopZ can only result from the dissociation of Cu(I) from CopZ into bulk solution.Because association of Cu(I) with CopZ occurs rapidly, the rate limiting step of Cu(I) transfer is dissociation, and so the rate of transfer reports directly on the dissociation rate constant.A) Overlaid UV-visible absorbance spectra of 0.5 Cu(I)/CopZ and apo-His 6 CopZ acquired over 46 hr.B) Absorbance intensity at 265 nm monitored over 46 hr for 0.5 Cu/CopZ and apo-His 6 CopZ.Proteins were 60 µM in 100 mM MOPS, 100 mM NaCl, pH 7.5.Only small changes in absorbance were observed.At longer time periods, scattering due to protein precipitation occured and so Cu(I)-transfer could not be followed further, but it is clear that little or no transfer of copper occurred during the experiment.If it is assumed that the loss of A 265

Figure S4 .
Figure S4.Distinct UV-visible 265 nm absorbance intensity responses of CopZ and CopAab during titration with Cu(I).Plots of absorbance intensity at 265 nm against Cu(I)/protein ratio during titration of CopZ (40 µM) and CopAab (40 µM) with Cu(I).The different responses indicate the direction of absorbance change when apo-and Cu-loaded protein samples are mixed together.

Figure S5 .
Figure S5.Absorbance changes at 265 nm upon mixing of apo-CopZ with apo-CopAab.Control experiment in which apo-CopZ (60 µM) was mixed with apo-CopAab (40 µM) and changes in absorbance at 265 nm measured over the first 70 ms by stopped-flow.Proteins were in 100 mM MOPS, pH 7.5, temperature was 15 o C. Virtually identical data were obtained at 25 o C.

Figure S10 .
Figure S10.ESI-MS of CopZ/CopAab mixtures: CopZ and CopAab species.A) Deconvoluted mass spectra of samples following mixing of 0.5 Cu/CopZ with apo-CopAab.CopZ monomer and dimer, and CopAab monomer and dimer regions are shown, as indicated.B) As in A) except that apo-CopZ was mixed with 0.5 Cu/CopAab.Proteins were in 20 mM ammonium acetate, pH 7.4.

Figure S12 .
Figure S12.Species present in the mass spectra after copper transfer between CopZ and CopAab.Bar graph illustrating the relative intensity of each species present in the deconvoluted mass spectra of samples, as indicated.Data from Figures 3, S8 and S9.