Modulation of MagR magnetic properties via iron–sulfur cluster binding

Iron–sulfur clusters are essential cofactors found in all kingdoms of life and play essential roles in fundamental processes, including but not limited to respiration, photosynthesis, and nitrogen fixation. The chemistry of iron–sulfur clusters makes them ideal for sensing various redox environmental signals, while the physics of iron–sulfur clusters and its host proteins have been long overlooked. One such protein, MagR, has been proposed as a putative animal magnetoreceptor. It forms a rod-like complex with cryptochromes (Cry) and possesses intrinsic magnetic moment. However, the magnetism modulation of MagR remains unknown. Here in this study, iron–sulfur cluster binding in MagR has been characterized. Three conserved cysteines of MagR play different roles in iron–sulfur cluster binding. Two forms of iron–sulfur clusters binding have been identified in pigeon MagR and showed different magnetic properties: [3Fe–4S]-MagR appears to be superparamagnetic and has saturation magnetization at 5 K but [2Fe–2S]-MagR is paramagnetic. While at 300 K, [2Fe–2S]-MagR is diamagnetic but [3Fe–4S]-MagR is paramagnetic. Together, the different types of iron–sulfur cluster binding in MagR attribute distinguished magnetic properties, which may provide a fascinating mechanism for animals to modulate the sensitivity in magnetic sensing.


Results
The binding of [2Fe-2S] and [3Fe-4S] in clMagR. Three conserved cysteines (C60, C124, and C126) of clMagR in a CX n CGC sequence motif (n is 63-65 in most cases) play critical roles in iron-sulfur cluster binding 18 (Fig. 1a), which has been further validated by alanines substitution mutant clMagR 3M (C60A, C124A, and C126A mutation of clMagR WT ). Strep-tagged clMagR WT and clMagR 3M were freshly prepared (labeled as "as-isolated") and purified to homogeneity under aerobic conditions. The clMagR WT protein showed brown color and clMagR 3M appeared colorless in the solution, indicating the presence or absence of iron-sulfur cluster, respectively. Consistently, the Ultraviolet-visible (UV-Vis) spectrum of as-isolated clMagR WT showed absorption from 300-to-600-nm region, and with an absorption peak at 325 and 415 nm, and a shoulder at 470 nm, whereas these absorption peaks were abolished in clMagR 3M (Fig. 1b). Circular dichroism (CD) spectroscopy was applied to characterize the types of iron-sulfur cluster and their protein environments during cluster maturation [42][43][44] . As shown in Fig. 1c, clMagR WT shows distinct positive peaks at 371 nm and 426 nm and three negative peaks at 324 nm, 396 nm, and 463 nm, respectively, suggesting the presence of [2Fe-2S] cluster 45 . However, it is worth pointing out that [4Fe-4S] or [3Fe-4S] clusters usually exhibit negligible CD intensity compared to [2Fe-2S] as shown previously in Nif IscA 45,46 , thus CD spectroscopy cannot exclude the existence of [4Fe-4S] or [3Fe-4S]. Electron paramagnetic resonance (EPR) spectroscopy was then used to analyze different states of as-isolated clMagR WT . The oxidized clMagR WT was S = 1/2 species, characterized by a rhombic EPR signal with g values at g 1 = 2.016, g 2 = 2.002, and g 3 = 1.997 (Fig. 1d) which disappeared at 45 K, suggesting the presence of [3Fe-4S] 1+ cluster 47,48 . After reduced with sodium dithionite (Fig. 1e), EPR signal from [2Fe-2S] cluster can be observed until the temperature increased to 60K [49][50][51] . Thus, two distinct iron-sulfur clusters were assigned by EPR spectroscopy of clMagR WT .

The assembly and conversion of [2Fe-2S] and [3Fe-4S] in clMagR.
Iron-sulfur cluster assembly of IscA, an clMagR homology protein in bacteria, is mediated by cysteine desulfurase IscS 2 . To elucidate how iron-sulfur cluster assembles in clMagR, time-course experiment was performed, and UV-Vis absorption and CD spectrum were used to monitor the IscS-catalyzed iron-sulfur cluster assembly in clMagR (Fig. 2). No signal of the iron-sulfur cluster was recorded when the reaction begins (0 min), and then the characteristic visible absorption peak and CD spectrum of clMagR WT appeared after 5 min, indicating [2Fe-2S] cluster assembled. As the reaction proceeds, the UV-Vis absorption intensity increased and after 180 min the signal was dominated by a broad shoulder centered at 415 nm (Fig. 2a). Concomitantly, the CD spectrum of the [2Fe-2S] center decreased and then almost disappeared after 180 min, indicating that [2Fe-2S] had been converted to [3Fe-4S] clusters and the reconstitution finished (Fig. 2b).
Iron-sulfur cluster assembly can be achieved by chemical reconstitution as well, since iron-sulfur apoproteins are able to spontaneously form iron-sulfur clusters in vitro when supplied with iron and sulfide under reducing conditions 1,43,57 . With this approach, started with apo-clMagR WT , we successfully reconstituted [3Fe-4S] cluster in clMagR protein, confirmed by UV-Vis absorption and CD spectrum result (Fig. 2c,d). To further validate if [3Fe-4S] is the sole type of iron-sulfur cluster in clMagR after chemical reconstitution, EPR and lowtemperature Resonance Raman spectroscopy were applied (Fig. 2e,f). The chemically reconstituted clMagR WT was S = 1/2 species, characterized by a rhombic EPR signal with g values at g 1 = 2.017, g 2 = 2.002, and g 3 = 1.994 (Fig. 2e). The signal is assigned to a S = 1/2 [3Fe-4S] 1+ cluster. The Low-temperature Resonance Raman spectrum showed an intense band at 346 cm −1 and additional bands at 406 and 420 cm −1 , which demonstrated that chemically reconstituted clMagR WT only contains [3Fe-4S] 1+ cluster (Fig. 2f).
We further investigated if clMagR could serve as an iron-sulfur carrier protein to accept [2Fe-2S] cluster from scaffold protein such as IscU 58 . Briefly, 400 µM holo-IscU was mixed with 400 µM strep-tagged apo-clMagR WT (d, e) X-band EPR spectrum of as-isolated clMagR WT at oxidized (d) and reduced status (e). The samples were frozen in TBS buffer and the spectrums were recorded at various temperatures (10 K, 25 K, 45 K, 60 K). (f) Lowtemperature resonance Raman spectra of as-isolated clMagR WT . Spectra were recorded at 17 K using 488 nm laser excitation. www.nature.com/scientificreports/ and incubated for 180 min under reduced condition, then, after desalting and strep-tactin affinity column separation, UV-Vis absorption and CD spectroscopy were applied the iron-sulfur cluster transfer process (Fig. 3a). The intensity of UV-Vis spectrum decreased in IscU ( Fig. 3b) but significantly increased in clMagR after reaction (Fig. 3d), indicating [2Fe-2S] cluster was transferred from IscU to clMagR 59 . Consistently, CD spectrum of IscU and clMagR also confirmed that [2Fe-2S] transfer occurred between IscU and clMagR (Fig. 3c,e). The resulting spectrum is very similar to that of the [2Fe-2S] intermediate assembled on IscS mediated reconstituted apo-clMagR (Fig. 2b).
Cys-60 is essential for clMagR to bind [3Fe-4S] cluster, not [2Fe-2S] cluster. Three conserved cysteines (C60, C124, and C126) of clMagR play critical roles in iron-sulfur cluster binding, and the substitute mutation of these three residues abolished iron-sulfur binding (Fig. 1b,c) 18 . To elucidate if three cysteines bind [2Fe-2S] and [3Fe-4S] differently, single Cys-to-Ala substitutions (C60A, C124A, and C126A) were made and their iron-sulfur binding properties were characterized. Freshly purified as-isolated clMagR C60A showed light brown color, and [2Fe-2S] cluster binding was verified by UV-Vis absorption and CD spectrum (Fig. 4a,b). A typical protein-bound [2Fe-2S] cluster absorption peak IscS-mediated iron-sulfur cluster assembly on clMagR monitored as a function of time by UV-Vis absorption (a) and CD spectroscopy (b). The spectra shown were taken with samples of pretreated clMagR to remove iron-sulfur clusters before reconstitution (apo-clMagR, 0 min, light green), incubated with IscS after 5 min (green), and after 180 min (dark green). (c, d) chemical reconstitution-mediated iron-sulfur cluster assembly on clMagR monitored as a function of time by UV-Vis absorption (c) and CD spectroscopies (d). The spectra shown were taken with samples of pretreated clMagR to remove iron-sulfur clusters before reconstitution (apo-clMagR, light green) and chemically reconstituted clMagR (chem re clMagR, purple). (e) X-band EPR spectrum of chemically reconstituted clMagR WT . The spectrum was recorded at 10 K. (f) Low-temperature resonance Raman spectra of chemically reconstituted clMagR. Protein and reagent concentrations are described in the Methods. Spectra were recorded at 17 K using 488 nm laser excitation. In contrast, purified as-isolated clMagR C124A and clMagR C126A were colorless, and the binding of iron-sulfur clusters was barely detectable by UV-Vis and CD spectrum ( Fig. 4c-f, light purple, and light blue lines, respectively). However, chemical reconstitution successfully reconstituted [3Fe-4S] cluster binding in both clMagR C124A and clMagR C126A (Fig. 4c-f, purple and blue lines, respectively). After chemical reconstitution, the UV-Vis absorption of both clMagR C124A and clMagR C126A mutants showed the signal of iron-sulfur cluster binding (Fig. 4c,e). Parallel CD spectrum studies confirmed both chemically reconstituted clMagR C124A and clMagR C126A have [3Fe-4S] cluster binding (Fig. 4d,f)  The UV-Vis absorption (b) and CD spectra (c) of IscU. IscU protein samples were taken before mixing with apo-clMagR (holo-IscU, black lines) and after incubated with apo-clMagR for 180 min (pink lines). (d, e) The UV-Vis absorption (d) and CD spectra (e) of clMagR. clMagR samples were taken before mixing with holo-IscU (apo-clMagR, light green lines) and after incubated with holo-IscU for 180 min (holo-clMagR, brown lines).  Chemical reconstitution-mediated iron-sulfur cluster assembly on apo-clMagR C60A monitored by UV-Vis absorption (a) and CD spectroscopies (b). The samples of spectra shown are as-isolated clMagR C60A (light orange) and chemically reconstituted clMagR C60A (chem re clMagR C60A , orange). (c, d) chemical reconstitution-mediated iron-sulfur cluster assembly on clMagR C124A monitored by UV-Vis absorption (c) and CD spectroscopies (d). The samples of spectra shown are as-isolated clMagR C124A (light purple) and chemically reconstituted clMagR C124A (chem re clMagR C124A , purple). (e, f) chemical reconstitution-mediated iron-sulfur cluster assembly on pigeon clMagR C126A monitored by UV-Vis absorption (e) and CD spectroscopies (f). The samples of spectra shown are as-isolated clMagR C126A (light blue) and chemically reconstituted clMagR C126A (chem re clMagR C126A , blue). SDS-PAGE results were shown in the right of corresponding UV-Vis spectra as inserts (a, c, e). The theoretical mass of the clMagR C60A monomer, clMagR C124A monomer and clMagR C126A monomer were 16.38 kDa. (g, h) The UV-Vis absorption (c) and CD spectra (d) of clMagR C60A obtained by mixing apo-clMagR C60A and holo-IscU which was recorded before the addition of apo-clMagR C60A (dotted orange lines) and after incubation with apo-clMagR C60A for 180 min (orange lines). Protein and reagent concentrations are described in the Experimental procedures. www.nature.com/scientificreports/ Considering clMagR can act as a carrier protein to accept iron-sulfur cluster from IscU (Fig. 3), it is worth testing if three cysteines play a different role in this process as well. Holo-IscU was mixed with apo-clMagR single cysteine mutants in a reduced state for 180 min. The apo status of all three mutants (labeled as apo-clMagR C60A , apo-clMagR C124A , and apo-clMagR C126A ) had no iron-sulfur cluster binding before mixing with holo-IscU, as shown by negligible UV absorption and CD intensities (Fig. 4g,h and Supplementary Fig. 1a-d, dotted lines). After incubation with holo-IscU and separation of IscU and clMagR mutants, clMagR C60A showed distinct changes in UV-Vis absorption and CD spectrum (Fig. 4g,h). The UV-Vis absorption increased and showed better-resolved peaks at 322 nm, 410 nm, 504 nm (Fig. 4g, orange line), and parallel CD spectra had distinct positive peaks (319 nm, 355 nm, 445 nm, and 534 nm) and four negative peaks (333 nm, 392 nm, 477 nm, and 579 nm, Fig. 4h), indicating [2Fe-2S] cluster was transferred from IscU to clMagR C60A . Interestingly, clMagR C124A and clMagR C126A could also accept [2Fe-2S] cluster transferred from holo-IscU, though the binding efficiency is much lower than clMagR WT and clMagR C60A , as verified by UV-Vis and CD spectrum ( Supplementary Fig. 1a-d). It seems that clMagR C60A accept [2Fe-2S] cluster from scaffold protein IscU more effectively compared with clMagR C124A and clMagR C126A . And after incubation with clMagR mutants, UV-Vis absorption of IscU significantly decreased, confirmed that iron-sulfur cluster transfer occurred in between holo-IscU and three clMagR mutants ( Supplementary Fig. 1e).
Again, our data demonstrated that three conserved cystines of clMagR played different roles on the iron-sulfur cluster binding, and especially Cys-60 is essential for clMagR to bind [3Fe-4S] cluster, not [2Fe-2S] cluster. Therefore, it is possible to obtain a [2Fe-2S] cluster binding only clMagR by mutating Cys-60. Thus, we labeled clMagR protein samples based on their iron-sulfur cluster in later experiments. For example, we labeled the chemically reconstituted clMagR WT as [3Fe-4S]-clMagR WT , and clMagR C60A that accepted [2Fe-2S] cluster from holo-IscU as [2Fe-2S]-clMagR C60A , to investigate the magnetic property of clMagR when it binds different iron-sulfur clusters.

[3Fe-4S]-clMagR shows different magnetic properties from [2Fe-2S]-clMagR. MagR has been
reported as a putative magnetoreceptor and exhibits intrinsic magnetic moment experimentally and theoretically when forms complex with cryptochrome (Cry) 18,20,21 . To elucidate if different iron-sulfur clusters binding in clMagR have different magnetic features and respond to external magnetic fields differently, we obtained [3Fe-4S] and [2Fe-2S] bound only clMagR protein by chemical reconstitution of clMagR WT (as [3Fe-4S]-clMa-gR WT ) and holo-IscU incubated and re-purified clMagR C60A (as [2Fe-2S]-clMagR C60A ), respectively, and measured the magnetic moment of these proteins with Superconducting Quantum Interference Device (SQUID) magnetometry. SQUID is a highly sensitive magnetometry to measure extremely subtle magnetic fields and to study the magnetic properties of a range of samples, including extremely low magnetic moment biological samples. Therefore, it has been regularly used as a first test to identify the specific kind of magnetism of a given specimen, such as ferromagnetic, antiferromagnetic, paramagnetic or diamagnetic, by measuring at different temperatures and external magnetic field strength. For example, B-DNA was identified as paramagnetic under low temperature by SQUID 60 .
Purified clMagR 3M was utilized as a control since it had no iron-sulfur cluster binding due to lack of cysteine residues (Fig. 1b,c). The magnetic measurement was done at different temperatures (5 K and 300 K) and MH curves (magnetization (M) curves measured versus applied fields (H)) were generated for three proteins to reflect the protein magnetic anisotropy. The MH curves of clMagR 3M clearly exhibited diamagnetic property at both 5 K and 300 K, suggesting that magnetism of clMagR is dependent on the iron-sulfur cluster (Fig. 5a,b, red lines). In contrast, [3Fe-4S]-clMagR WT showed superparamagnetic behavior at 5 K which has saturation magnetization (M S ) at 2 T about 0.22771 emu/g protein (Fig. 5a, purple line), [2Fe-2S]-clMagR C60A is paramagnetic at 5 K (Fig. 5a, orange line). Interestingly, at higher temperature such as 300 K, [2Fe-2S]-clMagR C60A is diamagnetic www.nature.com/scientificreports/ while [3Fe-4S]-clMagR WT is paramagnetic (Fig. 5b, orange line and purple line). The different magnetism, as well as the different saturation magnetization of clMagR with different iron-sulfur binding, are clearly important features of this putative magnetoreceptor, and worth further investigation and validation in vivo in the future.

Discussion
MagR (IscA), an A-type iron-sulfur protein MagR (IscA1), has been proposed as a candidate magnetoreceptor in the biocompass model 18 . Twenty MagR helically assembles as a rod-like polymer, surrounded by photo-sensitive cryptochrome (Cry), shows magnetic moment of roughly 0.09-0.1 µB/f.u in vitro. However, the mechanism of MagR/Cry complex to respond to external magnetic fields remain largely unknown. The characterization of iron-cluster binding of MagR would provide us clues to understand the currently unresolved mechanism. MagR (IscA) is a highly conserved iron-sulfur protein widely distributed across all major phyla. The characterization of iron-sulfur cluster binding in MagR in homing pigeon has not been fully investigated yet. Anaerobically purified of Azotobacter vinelandii Nif IscA was containing one [2Fe-2S] cluster per homodimer, while NifS-mediated reconstituted Nif IscA contains [4Fe-4S] cluster 46 . As-isolated yeast IscA1 was an apo-protein that could bind an [2Fe-2S] only after chemical reconstitution 59  Taking together, the characterization and identification of two forms of iron-sulfur cluster binding in pigeon MagR, and the observed distinguished magnetic features when MagR hosts different iron-sulfur clusters, suggested a possible dedicated regulatory mechanism of animal magnetoreception. Animals may utilize different iron-sulfur clusters as a magnetic switch to modulate the magnetic property and sensitivity of its magnetoreceptor during navigation. The study presented here extended our understanding of MagR's functional roles not only as an iron-sulfur protein but also as a candidate magnetoreceptor.

Method
Protein expression and purification. The expression vector containging MagR gene of the homing pigeon was constructed as described previously (Qin, Nature Materials, 2016), and the genes of clMagR C60A , clMagR C124A , clMagR C126A , and clMagR 3M were synthesized and cloned into the expression vector and expressed in E. coli strain BL21 (DE3), respectively. Bacteria cells were harvested after induction with 20 µM isopropyl -D-1-thiogalactopyranoside (IPTG) overnight at 288 K. And then lysed by sonication on ice and resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, pH 8.0) with complete protease inhibitor cocktail. After centrifugation, the supernatant was collected and loaded onto the Strep-Tactin affinity column (IBA). The column was washed about 20 column volumes (CV) with buffer W (20 mM Tris, 500 mM NaCl, pH 8.0) to remove unbound proteins. After washing, clMagR protein or its mutants were eluted from the Strep-Tactin affinity columns using buffer E (20 mM Tris, 500 mM NaCl, 5 mM desthiobiotin, pH 8.0). For all SDS-PAGEs, PageRuler Prestained Protein Ladder (Thermo Scientific, Product# 26616) was used as the molecular weight standards.
IscU sequence was obtained from NCBI (https:// www. ncbi. nlm. nih. gov/ gene/ 947002) and synthesized, then cloned into expression vector as mentioned previously with a His-tag fused on the N-terminal, and expressed in E. coli strain BL21 (DE3). Bacteria cells were harvested after induction with 20 µM IPTG overnight at 288 K, and then lysed by sonication on ice and resuspended in lysis buffer (20 mM Tris, 150 mM NaCl, 10 mM Imidazole, pH 8.0) with complete protease inhibitor cocktail. After centrifugation, the supernatant was collected and loaded onto the Ni-NTA affinity column (QIAGEN). The column was washed about 20 column volumes (CV) with buffer W (20 mM Tris, 150 mM NaCl, pH 8.0) to remove unbound proteins. After washing the matrix, proteins were eluted from the Ni-NTA matrix using elution buffer (20 mM Tris, 150 mM NaCl, 300 mM Imidazole, pH 8.0). www.nature.com/scientificreports/ The expression vector of IscS (pET 28-IscS) was a gift from Dr. Huangeng Ding's Lab. Bacteria cells were harvested after induction with 400 µM IPTG overnight at 288 K, and then lysed by sonication on ice and resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM Imidazole, pH 8.0) with complete protease inhibitor cocktail. After centrifugation, the supernatant was collected and loaded onto the Ni-NTA affinity column (QIAGEN).
The column was washed about 20 column volumes (CV) with buffer W (20 mM Tris, 500 mM NaCl, pH 8.0) to remove unbound proteins. After washing, IscS protein was eluted from the Ni-NTA matrix using elution buffer (20 mM Tris, 500 mM NaCl, 300 mM Imidazole, pH 8.0). Protein concentration was estimated using a nanodrop spectrophotometer (Thermo Fisher Scientific) with the target protein MW and coefficient of molar extinction ε retrieved from protparam tool website (https:// web. expasy. org/ protp aram/). Spectroscopic studies on iron-sulfur cluster binding in MagR. Different spectroscopic approaches were applied to study the iron-sulfur cluster binding in MagR: UV-visible (UV-Vis) absorption, Circular Dichroism (CD), Electron Paramagnetic Resonance (EPR), and low-temperature Resonance Raman (RR) spectroscopy.
Circular dichroism (CD) is a classic and robust method to either evaluate secondary structures in protein in the far UV range (190 to 260 nm) or to monitor protein-bound co-factors such as metals or iron-sulfur clusters in the near UV-visible range (300 to 600 nm) (Kelly, Current Protein and Peptide Science, 2000). Purified wildtype MagR protein and mutants were prepared at 4 mg/mL in TBS buffer (20 mM Tris, 150 mM NaCl, pH 8.0) and were measured in Circular Dichroism Spectrometer MOS-500 (Biologic) at room temperature in 1 cm path quartz cells. Buffer was used as blank control. Data were shown using the ellipticity value in mDeg as measure by the spectrometer with blank subtracted.
X-band (∼ 9.6 GHz) EPR spectra were recorded using EMX plus 10/12 spectrometer (Bruker, Billerica, MA), equipped with Oxford ESR-910 Liquid helium cryostat. Briefly, 1 mM oxidized clMagR (as-isolated clMagR) and 1 mM chemically reconstituted clMagR (chem re clMagR) in TBS buffer (20 mM Tris, 150 mM NaCl, pH 8.0) in a total volume of 0.2 mL mixed with 0.05 mL Glycerol were used, respectively. Reduced clMagR was obtained by adding 10 mM Na 2 S 2 O 4 into clMagR protein samples. Then, the protein samples were transferred into a 4 mm diameter quartz EPR tube (Wilmad 707-SQ-250 M) and frozen in liquid nitrogen. EPR signals of oxidized clMagR and reduced clMagR were recorded at various temperatures (10 K, 25 K, 45 K, and 60 K). Parameters for recording the EPR spectra were typically 2 G modulation amplitude, 9.40 GHz microwave frequency, and 2 mW incident microwave power, sweep time was 19.2 s.
For low-temperature resonance Raman (RR) spectra, purified proteins were concentrated to ∼ 1 mM and frozen by lyophilization and placed on the surface of a Si wafer chip with a 90-nm-thick SiO2 on the top sealed in a helium-cooled cryogenic station (Montana Instruments) at 17 K. The RR spectra were collected in backscattering geometry using a Jobin-Yvon HR800 system equipped with a liquid nitrogen cooled charge-coupled detector. The excitation wavelength is 488 nm from an Ar+ laser and a grating with groove density of 1800/mm was used to achieve a spectral resolution of 0.53 cm −1 . A long working distance 50× objective was used to ensure a high signal-to-noise ratio of the measured Raman spectra.
For IscS-mediated iron-sulfur cluster assembly experiment, Apo-clMagR (as mentioned above) was incubated with IscS (1 µM), ferrous ammonium sulfate (1.6 mM), l-cysteine (20 mM), and DTT (5 mM) in the same buffer for 180 min at room temperature. UV-Vis absorption and CD studies of the time course of iron-sulfur cluster assembly were carried out by measuring the spectra at different time intervals on one sample at room temperature. Briefly, before the reaction begins, the 20% of the reaction mixture was taken out and desalted, labeled as sample after "0 min". Another 20% of the reaction mixture was taken out after 5 min, desalted, and labeled as sample after "5 min". Then, the remaining reaction mixture was desalted after 180 min, and labeled as sample after "180 min". Purification of cluster-bound clMagR to remove excess reagents was achieved by loading the reconstitution mixture onto the desalting column (PD 10, GE Healthcare, 17085101) pre-equilibrated with buffer (20 mM Tris, 150 mM NaCl, pH 8.0) and eluted with buffer (20 mM Tris, 150 mM NaCl, pH 8.0).
For chemical reconstitution experiment, apo-clMagR was incubated with chemical reconstitution mixture (1 mM ferrous ammonium sulfate, 1 mM sodium sulfide, and 5 mM DTT) for 180 min at room temperature. Then, desalted to isolate the clMagR protein from reaction mixture using desalting column PD-10 (GE Healthcare, 17085101), and labeled the protein sample as "chem re clMagR".
In vitro iron-sulfur cluster transfer. Holo-IscU was obtained by chemically reconstituted as-isolated IscU. Briefly, as-isolated IscU was incubated with reconstitution mixture (1 mM ferrous ammonium sulfate, 1 mM sodium sulfide, and 5 mM DTT) for 180 min at room temperature. Then desalted to isolate the protein from reaction mixture using desalting column PD-10 (GE Healthcare, 17085101), and and labeled as "Holo-IscU" in Fig. 3a.
The experimental procedure of iron-sulfur transfer between clMagR and holo-IscU was illustrated in Fig. 3a. Briefly, strep-tagged apo-clMagR protein (400 µM) was incubated with holo-IscU (400 µM) in TBS buffer (20 mM Tris, 150 mM NaCl, pH 8.0) after reduced with 5 mM DTT for 2 h. After reaction, two proteins were then separated by loading the reaction mixture contains strep-tagged clMagR and non-tagged IscU to a strep-tactin