research papers
Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives
aDepartment of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark
*Correspondence e-mail: ph@kemi.dtu.dk
Using synchrotron radiation (SR), the crystal structures of T6 bovine insulin complexed with Ni2+ and Cu2+ were solved to 1.50 and 1.45 Å resolution, respectively. The level of detail around the metal centres in these structures was highly limited, and the coordination of water in Cu site II of the copper insulin derivative was deteriorated as a consequence of radiation damage. To provide more detail, was used to improve the information level about metal coordination in each derivative. The nickel derivative contains hexacoordinated Ni2+ with trigonal symmetry, whereas the copper derivative contains tetragonally distorted hexacoordinated Cu2+ as a result of the Jahn–Teller effect, with a significantly longer coordination distance for one of the three water molecules in the coordination sphere. That the copper centre is of type II was further confirmed by (EPR). The coordination distances were refined from with standard deviations within 0.01 Å. The insulin derivative containing Cu2+ is sensitive towards when exposed to SR. During the reduction of Cu2+ to Cu+, the coordination geometry of copper changes towards lower coordination numbers. Primary damage, i.e. was followed directly by XANES as a function of radiation dose, while secondary damage in the form of structural changes around the Cu atoms after exposure to different radiation doses was studied by crystallography using a laboratory diffractometer. Protection against and subsequent radiation damage was carried out by solid embedment of Cu insulin in a saccharose matrix. At 100 K the was suppressed by ∼15%, and it was suppressed by a further ∼30% on cooling the samples to 20 K.
Keywords: bovine insulin; nickel; copper; X-ray absorption spectroscopy; EXAFS; XANES; EPR; photoreduction; radiation damage.
3D view: 4m4f,4m4h,4m4i,4m4j,4m4l,4m4m
PDB references: Cu insulin, 4m4f; 4m4h; 4m4i; 4m4j; 4m4l; Ni insulin, 4m4m
1. Introduction
Within the field of protein crystallography, an increased understanding of radiation damage from X-rays has developed throughout the last decade (Sliz et al., 2003; Ravelli & Garman, 2006; Garman & Nave, 2009). In particular, metal centres in metalloproteins are sensitive to radiation damage, and for redox-active proteins in which transition metals are actively involved it is crucial that the metal centres are accurately characterized for correct interpretation of the function of the protein.
In X-ray structures, ; Pearson & Owen, 2009; De la Mora-Rey & Wilmot, 2007). Among the spectroscopies used in combination with XRD are X-ray absorption spectroscopy (XAS; Arcovito & della Longa, 2012; Cotelesage et al., 2012; Yano & Yachandra, 2008; Strange et al., 2005; Hasnain & Strange, 2003), UV–visible absorption spectroscopy (Hersleth & Andersson, 2011; Ellis et al., 2008; Pearson et al., 2004) and Raman spectroscopy (Katona et al., 2007; McGeehan et al., 2011; Hersleth & Andersson, 2011).
of metal centres and the subsequent radiation damage is a well known problem when high-intensity synchrotron radiation (SR) is used. The detailed structure around a metal atom is often distorted owing to radiation damage, which means that looking for loosely bound water molecules in an active site, or determining the detailed coordination geometry of the metal, is not always possible from diffraction experiments, even at high resolution. Complementary techniques to X-ray diffraction (XRD) are thus required to extract information about the metal identity, ligation and redox states. The development of multifunctional beamlines which combine macromolecular crystallography with spectroscopy has facilitated single-crystal spectroscopy experiments on protein crystals with concurrent collection of crystallographic data (Antonyuk & Hough, 2011XAS is well suited for studying the redox states and ligation of metals, and includes both extended X-ray absorption fine structure (EXAFS) spectroscopy, which provides detailed information about the radial distribution of atoms, leading to precise determination of bond distances, and X-ray near-edge structure (XANES) spectroscopy, which primarily provides information about in situ studies of radiation damage. Absorption spectroscopy has previously been used for studying in metalloproteins, e.g. the putidaredoxin containing an [Fe2S2] cluster (Corbett et al., 2007), Fe-containing neuroglobin (Arcovito et al., 2008), the Mn-complex in photosystem II (Grundmeier & Dau, 2012), copper nitrite reductase (Hough et al., 2008) and myoglobin (della Longa et al., 2003). Direct radiation damage of selenomethione side chains has also been studied by XANES (Holton, 2007).
and connectivity, as the near-edge region is more dominated by multiple scattering events. Compared with the time for a diffraction experiment or the collection of a full spectrum, XANES experiments can be performed much more rapidly, which makes this technique advantageous for spectroscopic probing of andIn this work, we have studied the structures of Ni2+ and Cu2+ derivatives of bovine insulin by XRD and and the coordination geometry of Cu insulin by (EPR). Hexameric insulin binds two metal ions by the coordination of three histidine residues (HisB10) to each of the two metal ions. In its natural form the metal is zinc, but insulin is also known to have affinity towards other transition metals, including Mn, Fe, Co, Ni, Cu and Cd (Schlichtkrull, 1956), all in the +2 In the T6 conformation of insulin studied here, both of the metal sites are exposed to the solvent. The coordination sphere for each metal site is thus completed by water molecules, some of which are weakly coordinated and easily exchanged. The T6 insulin system thereby provides a well suited model for studying metal centres with labile water molecules. As Cu2+ is easily reduced by high-intensity X-ray radiation, the Cu insulin derivative furthermore represents a suitable model system for and following radiation damage. Owing to the unique spectral features of Cu+ at its X-ray the of Cu2+ to Cu+ can be followed by XANES as a function of the radiation dose delivered to the system. We present suggestions as to how can be minimized and follow the radiation-induced structural changes of the Cu sites by in-house XRD.
2. Experimental
2.1. Preparation of crystalline nickel and copper insulin
Single crystals as well as microcrystals of Ni2+ and Cu2+ insulin were prepared in analogy to the procedures for T6 Zn insulin described by Frankaer et al. (2012). Deviations from the reported procedures are reported in the following.
Nickel and copper insulin single crystals were grown using the vapour-diffusion technique. 2 µl of a solution consisting of 7.5 mg ml−1 metal-free insulin adjusted to pH 2.0 using aqueous HCl was mixed with 2 µl reservoir solution and equilibrated in a hanging drop against 1 ml reservoir solution with a composition of 0.05 M sodium citrate, 15%(v/v) acetone and 15 mM nickel(II) acetate or 7.5 mM copper(II) acetate, respectively. In the nickel and copper reservoirs, the pH was adjusted to 7.4 and 7.1, respectively, using aqueous HCl. After 5 d, crystals with dimensions of 200–400 µm were observed. Single crystals were cryoprotected as described by Frankaer et al. (2012) and mounted directly under a 100 K cryostream at the diffractometer before diffraction analysis.
Microcrystal samples of nickel and copper insulin were used for X-ray absorption spectroscopy measurements and were prepared using the method for the preparation of T6 Zn insulin microcrystals but with substitution of zinc(II) acetate with nickel(II) acetate or copper(II) acetate.
A powdered sample of Cu insulin microcrystals embedded in a saccharose matrix was obtained using the method described by Ascone et al. (2000). A slurry containing Cu insulin microcrystals and saccharose at a sucrose:insulin ratio of 3:1(w:w) was prepared by adding 1 ml 75 g l−1 sucrose solution to isolated Cu insulin microcrystals crystallized from 25 mg insulin. The slurry was rapidly frozen in 2-propanol/dry ice and lyophilized.
2.2. Single-crystal diffraction
Single-crystal diffraction data for Ni and Cu insulin were collected on beamline I911-2, MAX II at MAXIV Laboratory, Lund, Sweden using a MAR Research MAR165 CCD detector. The 12 photons s−1 mm−2. The scattering properties generally improved after annealing the crystals a few times. The data were processed and scaled using XDS and XSCALE (Kabsch, 2010).
used was estimated to be 1.0 × 10In-house single-crystal data from crystals exposed to different radiation doses were collected from Cu insulin crystals using an Agilent Supernova diffractometer. From a large well diffracting crystal, a complete data set to 1.9 Å resolution was collected (CuInsA; Table 1) while the radiation dose was kept at a minimum (0.01 MGy). High-dose data sets (CuInsB–D; Table 1) were successively collected on a smaller crystal diffracting to beyond 1.9 Å resolution using a longer exposure time. All in-house diffraction data were collected at 100 K using Cu Kα radiation (λ = 1.5419 Å) with a of 1.0 × 1010 photons s−1 mm−2 at the sample. The crystal sizes were carefully determined from photographs using the CrysAlisPro software (Agilent Technologies) and radiation doses were calculated by RADDOSE (Murray et al., 2004; Paithankar et al., 2009) and are listed in Table 1. Data were processed and scaled using the CrysAlisPro software (Agilent Technologies).
‡Rmerge is defined as , where 〈I(hkl)〉 is the mean intensity of a set of equivalent reflections. §The B-factor analysis was performed using BAVERAGE included in CCP4 (Winn et al., 2011). ¶The definition of the Ramachandran plot regions is according to Kleywegt & Jones (1996). ††R and Rfree = , where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree was calculated with a random 5% subset of all reflections that was excluded from the |
Using the peptide chain from the T6 Zn insulin structure (PDB entry 4e7t ; Frankarr et al., 2012) as a starting model, the structures were refined using REFMAC5 (Murshudov et al., 2011) and PHENIX (Adams et al., 2010). Model building and editing were carried out using WinCoot (Emsley et al., 2010). The structures were validated using PROCHECK (Laskowski et al., 1993), WHAT_CHECK (Hooft et al., 1996) and the structure-analysis server STAN (Kleywegt & Jones, 1996). Data-collection and for all crystals are summarized in Table 1.
2.2.1. Ni insulin synchrotron structure
Two Ni atoms were included, and the side chains of residues GlnB4.1, ValB12.1, LeuB17.1, CysA11.2 and GlnB4.2 were modelled in two alternate conformations. A total of 80 water molecules were inserted. PHENIX and H atoms were included. The atomic displacement factors for the peptide chain were refined by a combination of TLS and isotropic The TLS domains were as follows: residues 1–8 and 13–19 in the A chains, residues 9–18 in the B chains and a group containing residues 23–27 of two adjacent B chains. Other atoms were refined isotropically. Validation showed that only one residue, SerA9.1, was in the outlier region of the Ramachandran plot (as defined by Kleywegt & Jones, 1996).
was carried out in2.2.2. Cu insulin synchrotron structure
Two Cu atoms were inserted, and the side chains of residues GlnB4.1, ValB12.1, LeuB17.1, CysA11.2 and ValB12.2 were modelled in two alternate conformations. 98 water molecules were inserted in total and H atoms were included. The
procedure was analogous to that used for Ni insulin. Validation showed that only one residue, SerA9.1, was in the outlier region of the Ramachandran plot.2.2.3. Cu insulin in-house structures
The evolution of radiation damage was studied from comparison of four models corresponding to different values of absorbed dose from 0.01 to 0.30 MGy. It should be emphasized that the three data sets (CuInsB–D) were collected from the same crystal, which explains the similar unit-cell parameters and mosaicity values observed for these structures. In the subsequent structure ), and all structures were refined to a resolution of 1.9 Å, resulting in R factors below 0.17 and Rfree factors below 0.24. The residue SerA9.1 was in the outlier region in the Ramachandran plot for all four structures. The outlier region also included SerA9.2 in CuInsB–D and ProB28.1 in CuInsA.
two Cu atoms were included in each model. The C-terminal residue AlaB30.1 was disordered in all four structures and was therefore not included. PheB1.1 was not modelled in CuInsA and CulnsD. The side chains of residues ValB12.1 and CysA11.2 were modelled in two alternate conformations for all structures, and for CuInsB, CuInsC and CuInsD further alternate conformations were found for the side chains of residues GluB13.1 and ValB12.2. A number of water molecules ranging from 52 to 68 was included in each of the structures (see Table 12.3. X-ray absorption spectroscopy
Ni and Cu K-edge X-ray absorption spectra were recorded on beamline I811 at the synchrotron at MAXIV Laboratory, Lund, Sweden (Carlson et al., 2006) using a Si(111) double-crystal monochromator detuned 60% at 9333 and 9829 eV for Ni and Cu insulin, respectively. The samples were mounted in 1 mm thick sample holders (Frankaer et al., 2011) and were cooled to either 20 or 100 K in a cryostat using liquid helium or liquid nitrogen, respectively. Fluorescence data were collected using a PIPS PD-5000 (passivated implanted planar silicon) detector from Canberra with the scan ranges and times listed in Table 2. In order to ensure that no radiation damage of the sample had taken place, or at least that it was minimized, a fast scan was performed after the collection of each spectrum. For Cu insulin the sample was renewed between each scan.
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The following reduction and analysis of WinXAS (Ressler, 1998) and EXCURVE (Gurman et al., 1984, 1986; Binsted et al., 1991) in accordance with the procedure described by Frankaer et al. (2012). Calculation of XANES spectra by finite-difference methods (FDM) was performed using FDMNES (Joly, 2001).
data were carried out usingThe RADDOSE (Murray et al., 2004; Paithankar et al., 2009) are summarized in Table 2. The XANES spectra were energy-calibrated using an internal Cu foil reference sample and were background-subtracted and normalized using ATHENA (Newville, 2001; Ravel & Newville, 2005). The signal from copper in +1 was extracted from the peak located at 8983 eV (Kau et al., 1987). This peak was isolated by the subtraction of a fast-scan XANES spectrum collected at 20 K, in which no peak was observed at 8983 eV and which hence was obtained before takes place. The area under the isolated peak was calculated by fitting a Gaussian function to the left-hand side of the peak, since the right-hand side is difficult to resolve owing to its location very close to the absorption edge.
of Cu insulin at different temperatures with and without saccharose protection was monitored by XANES. The XANES data-collection specifications and dose calculations as performed by2.4. Electron paramagnetic resonance
EPR was recorded on solid microcrystalline Cu insulin. The sample was recorded at room temperature (RT) and at 77 K using a liquid-nitrogen finger dewar in the ST4102 resonator of an X-band Bruker EMX EPR spectrometer. The microwave frequency was 9.34 GHz, the microwave power was 5 mW, the modulation frequency was 100 kHz and the modulation amplitude was 8 G. The spectrum was recorded over three sweeps. The spectrum of the empty tube was subtracted and the spectrum was fitted using the spin Hamiltonian-based program W95EPR (Neese et al., 1996).
3. Results
3.1. Nickel insulin
In the 6 conformation in analogy to the T6 zinc insulin structure (Frankaer et al., 2012). The structure contains two Ni2+ ions exposed to the solvent in both of the open T3 sites, as shown in Fig. 1. The Ni atoms are hexacoordinated, with the coordination sphere consisting of three equivalent imidazole N atoms and O atoms from three equivalent water molecules owing to the threefold symmetry. The distances between Ni and the O atoms of the water molecules are 2.12 and 2.23 Å in sites I and II, respectively.
of insulin co-crystallized with nickel, the hexamers were found to adopt the TThe extracted k3-weighted spectrum and the modulus of the phase-corrected Fourier transform of Ni insulin are presented in Fig. 2. The shape of the k3-weighted χ(k) for Ni insulin has a high resemblance to that of T6 Zn insulin reported by Frankaer et al. (2012), indicating an analogous pseudo-octahedral coordination. Distances and Debye–Waller factors, as optimized from a restrained using coordinates from the SR structure, are presented in Table 3 and the fit is shown in Figs. 2(a) and 2(b).
‡The φ angle (polar coordinates) was refined in order to allow some movement of the atoms in the restrained imidazole ring. |
A XANES spectrum calculated by the FDM method is shown in Fig. 2(c) using the coordinates from the model optimized by There is good agreement between the experimental and calculated spectra. The high-intensity white line is in agreement with the XANES spectra reported for other hexacoordinated nickel complexes (Colpas et al., 1991), thereby verifying the pseudo-octahedral coordination.
3.2. Copper insulin
Hexameric copper insulin is found to adopt the T6 conformation in all of the crystal structures reported here. The synchrotron of copper insulin contains two Cu ions coordinated by the HisB10 residues in both T3 sites, as shown in Fig. 3. In site I a hexacoordinated copper is observed with a Cu–water distance of 2.25 Å. In site II the coordination has a more tetrahedral character, in which one water molecule can be modelled in the first solvation shell on the threefold symmetry axis at 2.67 Å from the Cu atom, as shown in Fig. 3. However, the electron density is still reminiscent of a pseudo-octahedral coordination. Also, a weaker electron density is observed in the second solvation shell, which indicates deterioration of the water structure as a consequence of radiation damage.
3.2.1. Radiation damage monitored by XANES and in-house XRD
XANES spectra collected on Cu insulin at 100 K as a function of radiation dose (ranging from approximately 0.1 to 1.0 MGy) are shown in Fig. 4(a). As the radiation dose increases a peak arises at 8983 eV. This peak originates from the 1s→4p electronic transition of copper in +1 (Kau et al., 1987), thereby showing that of Cu2+ to Cu+ takes place. Furthermore, it is seen in Fig. 4(a) that the intensity of the white line decreases as takes place. This indicates a change in the coordination surroundings towards tetrahedral geometry (Kau et al., 1987).
In the crystal structures solved from data collected using our in-house equipment, in which the crystals were exposed to different radiation doses, the copper sites are shown in Fig. 5. The water molecules have been removed from the structures and difference maps have been calculated. At low radiation doses the difference maps indicate hexacoordinated copper in both copper sites, which is in agreement with the XANES results and analogous to the Ni insulin structure. At higher doses no significant changes in the coordination of the Cu I site are observed, whereas the Cu II site is seen to change with increasing radiation dose. This rearrangement is in agreement with the decrease in white-line intensity seen in the XANES spectra (Fig. 4a).
Generally, the water structure around the Cu sites was difficult to model. The water–water distances appear to be closer than normal hydrogen-bond distances, down to 2.0 Å. This problem could not be solved by decreasing the occupancy or increasing the lower cutoff for intermolecular water–water distances. In our structures no significant difference in the first water shell around the Cu I site was observed in the four different structures. In the second water shell, the water molecule next to the coordinating water apparently moves away from the Cu site with increasing radiation dose. The water–water distance also increases, from 2.00 to 2.35 Å, which indicates minor changes, in site I. However, these changes were only monitored and were not explained by the final models.
3.2.2. Minimizing photoreduction
The evolution of the (b). Cu+ formation is probed by the peak appearing at 8983 eV and the relative amount of Cu+ has been calculated by integration of this peak, as described in §2.3. The peak areas are plotted as a function of radiation dose/exposure time. As seen from this figure, the amount of Cu in +1 increases with increasing radiation dose. Comparing the three samples, it is seen that is slowed by approximately 15% by embedding the protein in a saccharose matrix and by a further 30% by cooling the saccharose-protected sample from 100 to 20 K.
in Cu insulin samples prepared without saccharose at 100 K, with saccharose at 100 K and with saccharose at 20 K is shown in Fig. 43.2.3. Coordination of Cu in an undamaged sample
The experimental EPR spectrum of microcrystalline Cu insulin is shown in Fig. 6 (black line). The experimental spectrum has been fitted (red line) with the usual axial spin Hamiltonian model used to model EPR spectra of Cu2+ (Neese et al., 1996), with g-values g∥ = 2.30 (2), g⊥ = 2.06 (2) and with the parallel component of the coupling constant to the nuclear spin of copper being A∥ = 480 (30) MHz [0.016 (1) cm−1]. The perpendicular component A⊥ was not resolved and therefore was not well determined, but was set to 30 MHz in the fitted spectrum shown. The line shape was Gaussian. The differences between the two copper sites and the coupling to the nitrogen nuclei were not resolved. The parameters correspond to a type II Cu2+ protein with two nitrogen donors and two oxygen donors (Peisach & Blumberg, 1974) and a predominantly tetragonal site geometry (Savelieff et al., 2008). This corresponds to Cu2+ being coordinated to two imidazole N atoms from histidine residues and to two water molecules in the plane of the dx2 - y2 orbital, with the final imidazole on the z axis.
For Cu2+ with six nitrogen or oxygen donors the trigonal symmetry will give rise to a degenerate ground state of Cu2+ owing to the d9 electronic structure. This will be Jahn–Teller unstable and therefore some geometric distortion is expected to take place. Nevertheless, in all crystal structures copper is bound in positions of C3 symmetry and coordinates to three equivalent histidine residues just as in the Ni2+ and Zn2+ analogues. EPR clearly demonstrates that in the low-dose crystal the actual experienced by the copper centre in both sites I and II is close to tetragonal and therefore one histidine residue must be different from the other two. In order to investigate the discrepancy between the crystallographic C3 and the EPR data, we proceeded to compare different geometry models with the data.
For k range 2.8–13.3 Å–1. The structural parameters of the optimized geometries are presented in Table 4. XANES spectra calculated from models as optimized by are presented in Fig. 7.
analysis, four different copper geometries were tested by building models with trigonal symmetric hexacoordination, tetragonally elongated hexacoordination, tetragonally distorted square-pyramidal geometry (pentacoordination) and trigonal symmetric pseudo-tetrahedral geometry (tetracoordination). Coordinates were taken from the SR and the presence and position of the O atoms from water were modified according to each of the four geometries. Before calculation of XANES spectra each model was optimized by a constrained in the
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As seen in the calculated XANES spectra in Fig. 7, the intensity of the white line decreases with decreasing The high-intensity white line observed in the experimental spectrum thus shows that copper is hexacoordinated. The poor fit to the pseudo-tetrahedral model further confirms the presence of type II copper sites. As seen in Table 4, it is noteworthy that the trigonal hexacoordinated model fits the data poorly, which is in agreement with the EPR results and with the general coordination preferences of Cu. Hence, the best fits from both the as well as the calculated XANES are obtained from the tetragonally elongated hexacoordinated model: Rexafs = 14.38% and Rxanes = 3.62%. The extracted k3-weighted spectrum and the modulus of the phase-corrected Fourier transform of Cu insulin are presented in Fig. 8, together with the fit for the tetragonally elongated hexacoordinated model. The optimized distances and Debye–Waller factors of this model are presented in Table 5. It is seen that all three distances from copper to nitrogen ligands in the histidine residues are very similar and that the tetragonal distortion is thereby most prominent for the axial water molecule, which is a further 0.2 Å away from Cu2+.
‡Average distances from Cu to histidine and water atoms of both Cu sites in the §The rotation angle of the histidine unit around an axis orthogonal to the imidazole plane passing through the N∊2 atom was included in the |
4. Discussion
4.1. General conformation
Hexameric insulin was successfully crystallized with divalent cations of nickel and copper, and the structures were solved. The T6 conformation was observed in all structures and the structures were compared with the zinc T6 structure and other structures in the PDB by superposing independent T2 dimers in SUPERPOSE (Krissinel & Henrick, 2004), in which the Cα displacements were minimized and the root-mean-square deviations (r.m.s.d.s) were calculated. Both structures show high resemblance to the bovine zinc T6 insulin structures deposited in the PDB [PDB entries 2a3g (Smith et al., 2005) and 4e7t (Frankaer et al., 2012)], with an r.m.s.d. below 0.3 Å. In analogy to bovine zinc T6 insulin, comparison with the structures of human Ni insulin and Cu insulin [PDB entries 3exx (Prugovečki et al., 2009) and 3tt8 (Prugovečki & Matković-Čalogović, 2011)] show larger discrepancies (r.m.s.d.s around 1.3 Å) owing to a different conformation of the B1.2–B3.2 chain. The weak determination of the B1.2–B3.2 residues may be a consequence of a partially disordered N-terminus of the B chain.
4.2. Coordination of nickel in Ni insulin
The octahedral coordination of nickel in insulin observed in the w1 distances are slightly longer in the (2.18 Å on average) compared with the distance as refined by (2.10 Å). Similar deviations were observed between the and the results for T6 Zn insulin (Frankaer et al., 2012), which was explained by the higher radiation doses in the diffraction experiment. The observed nickel coordination is generally in very good agreement with the human Ni insulin structure (PDB entry 3exx ; Prugovečki et al., 2009).
is in good agreement with the results obtained by In general, the Ni–O4.3. of copper in Cu insulin
The sensitivity to 2+ is very stable, Cu2+ can easily be reduced to Cu+ in the X-ray beam. The problem with of copper centres is well known and preservation by lyophilizing protein solutions in saccharose has previously been reported for haemocyanin and haemoglobin (Ascone et al., 2000). By keeping the protein in a solid phase, the mobility of damaging species is reduced because free diffusion is hindered.
is first and foremost dependent on the metal coordinated to the protein. Whereas NiAs demonstrated in Fig. 4(b), is suppressed when the Cu insulin crystals are embedded in a saccharose matrix and is even further reduced on cooling to 20 K (by approximately 40% in total). The experiments showed that this preservation technique also can be performed on microcrystalline samples, which makes this method even more versatile.
The characteristic feature in the XANES spectrum from Cu in φ. As seen from the successive collected XANES spectra, takes place immediately after exposure. Compared with the evolution of the electron density in the in-house crystal structures (Fig. 5), the structural changes around copper in site II seem to be detectable at radiation doses of around 0.1 MGy and above. Thereby, the radiation damage to the water coordination could be initiated by the of Cu2+ to Cu+.
+1 makes it possible to suggest a mechanism by which the photodegradation proceeds as a function of radiation doseThe + over the entire series is shown in Fig. 9 (circles). As seen from the figure, the reaction does not seem to reach equilibrium within the first 5 h. Instead, Cu+ builds up at an approximately constant rate after approximately 1 MGy. A similar trend has been observed for the reduction of Fe3+ in putidaredoxin after long exposures (doses of up to 12 MGy) by Corbett et al. (2007). Although exponential curves seem to accurately reproduce the data in the low-dose range (<1 MGy), it is noteworthy that the rate at which the reduced species build up seems to be linear after doses exceeding 1 MGy. This suggests a pre-equilibrium mechanism (Rae & Berberan-Santos, 2004) following the scheme shown in Fig. 10.
was monitored for approximately 300 min for a Cu insulin sample embedded in saccharose at 20 K and the formation of CuIn Fig. 10 a reversible redox reaction between the hexacoordinated tetragonally distorted copper insulin species A and B is followed by a step in which the water structure is deteriorated: species C. Following this reaction as a function of radiation dose φ, rate constants k1, k2 and k3 were determined from a numerical solution of the differential equation system
This resulted in k1 = 5.9 MGy−1, k2 = 0.3 MGy −1 and k3 = 1.2 MGy−1. The concentrations of B and C, and the total amount of copper in +1, [B + C], are shown individually in Fig. 9. The high value of k1 compared with k2 shows that the equilibrium between Cu2+ and Cu+ is shifted to the right. The rearrangement of the water structure in B and the subsequent relaxation is slower and acts as a way to stabilize the Cu+ centres, thereby dragging the pre-equilibrium to the right.
4.4. Coordination of copper
The EPR results presented here are in excellent agreement with previous Cu insulin EPR results by Brill & Venable (1968), who found that the two Cu sites had identical geometry. Similar type II Cu2+ complexes involving histidine ligands have been observed in recent EPR studies of Cu2+-containing amyloid-β (Shin & Saxena, 2008; Jun et al., 2009). Comparison with other biological copper(II) complexes, such as Cu–salicylate complexes (Valko et al., 1990), shows good agreement with tetragonal geometry with two N and two O atoms in the equatorial plane.
As previously shown, Cu insulin is very sensitive to A in Fig. 10) is in agreement with the results from the non-destructive EPR experiment, the low-dose X-ray experiments (in-house XRD) and the low-temperature (saccharose-protected samples at 20 K). The distances from Cu to the N atoms of histidine residues are generally longer in crystal structures (2.10 Å on average) compared with the distances as refined by (1.98/2.02 Å) and, in analogy to the nickel structure, a similar trend is observed for the copper–water distances. In general, the Cu-ligation distances determined in this study fall well within the range of both Cu–N and Cu–water distances observed in other protein structures containing copper type II centres (Abriata, 2012), and the hexacoordinated coppers observed in the low-dose structures are in good agreement with the coordination of copper in the human Cu insulin structure (PDB entry 3tt8 ; Prugovečki & Matković-Čalogović, 2011).
and subsequent radiation damage of the Cu centres. Hence, the coordination of copper as determined by the different X-ray techniques depends on the radiation dose. A tetragonally distorted hexacoordinated geometry of copper in both sites (speciesWhereas an accurate characterization of the copper ligation is excellently provided by 6 insulin with zinc (PBD entry 1mso ; Smith et al., 2003) as well as the 1.12 Å resolution structure of human Cu insulin (PDB entry 3tt8 ; Prugovečki & Matković-Čalogović, 2011) reveals similar unrealistically short water–water distances close to the metal sites.
and EPR in combination with XRD, the information about water coordination in the second solvation shell is limited using these techniques. For and EPR the limiting factor is the spectral resolution, whereas for XRD the limit is determined by the degree of radiation damage, which induces structural changes around the metal atoms. As illustrated by the crystal structures presented here, a chemically reasonable model is difficult to obtain as the intermolecular water distances are unrealistically low. This seems to be a general problem. Comparison with other high-resolution insulin structures such as the 1.0 Å resolution structure of human TIn theory, the tetragonally distorted copper coordination of Cu2+ is in conflict with the C3 symmetry observed in the Cu insulin crystal structures, as it will cause the distance from Cu to one of the three histidines as well as to one of the water molecules to differ from the other two. The elongation is more expressed in the Cu–water bond since the water molecules are less restricted. Nevertheless, if the tetragonal elongation is equally distributed among, or resonating between, the three symmetry-related N—Cu—O axes, the average effect would not break down the crystallographic C3 symmetry of the metal site.
5. Conclusions
The coordination of Cu and Ni in bovine insulin derivatives was studied by combining 2+ insulin derivative clearly revealed the presence of type II copper sites in which copper adopts a tetragonal coordination. We have demonstrated that crystallography must be complemented by other techniques if structural details are to be resolved around the metal sites, in particular for labile ligated sites such as the water molecules present in the insulin T6 conformation studied here. To some extent the sensitivity towards radiation damage from the X-radiation depends on the actual ligation of a metal, but it primarily depends on the type of metal and its Nickel insulin containing octahedrally coordinated Ni2+ was found to be stable throughout the X-ray experiments, whereas the Cu2+ in copper insulin suffered from in which Cu+ was formed and the coordination sphere was disrupted.
with crystallography. Both nickel and copper were found to be hexacoordinated and the distances between the metal and its ligands were very precisely determined using Furthermore, EPR measurements of the CuThe importance of efficient protection against radiation damage was illustrated by following the
(primary damage) by XANES as a function of radiation dose and by monitoring the structural changes (secondary damage) around copper at different radiation doses by crystallography using an in-house X-ray source. At 100 K, disruption of the water structure in Cu site II was detected at doses above 0.1 MGy. We found that the could be supressed by approximately 15% by embedding the protein in a saccharose matrix and by a further 30% by cooling the saccharose-protected sample to 20 K. This study thus recommends the use of the solid saccharose matrix-embedment protocol and liquid helium-based cooling for studying photoreduction-active metals in biological systems.Supporting information
3D view: 4m4f,4m4h,4m4i,4m4j,4m4l,4m4m
PDB references: Cu insulin, 4m4f; 4m4h; 4m4i; 4m4j; 4m4l; Ni insulin, 4m4m
Acknowledgements
We gratefully acknowledge the Carlsberg Foundation for funding the laboratory diffractometer used in some of these experiments. SM acknowledges The Danish Independent Research Council, Technology and Production for financial support. Other portions of this research were carried out on beamlines I811 and I911 at MAXIV Laboratory synchrotron-radiation source, Lund University, Sweden. We acknowledge the work of Katarina Norén in assistance with
experiments on beamline I811 and for helpful discussions throughout the project. Financial support was provided by the Danish National Research Council through the Danscatt program.References
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