In favorable cases, the degree to which the structures for a complementary pair of chimeric protein hybrids correspond to a simple sum of the parental protein structures can be characterized via a differential analysis of the chemical shifts and NOE intensities. Although quantitative prediction of chemical shifts presents a major ongoing challenge, the exquisite sensitivity of these shifts to the local environment can provide an excellent criterion by which to identify the boundary junctions to be used in construction of an initial protein model and, subsequently, for qualitative assessment of that model. Quantitative NOE peak intensities provide an independent criterion against which the consistency of the structure of the hybrid proteins with the corresponding "cut-and-paste" models can be assessed. The more straightforward dependencies of the NOE cross relaxation rate upon the local geometry and internal motion provide a basis for structural interpretation of the intensity differences that occur between the NOE crosspeaks of the hybrid and the corresponding parental spectra as predicted from the 'cut-and-paste" model.

In the analysis of whether the two metal binding site-swapped hybrids can each be accurately represented as a sum of the parental Pf and Cp rubredoxin structures, the critical decisions in the initial model construction involve those residues that are conserved in each sequence but may differ in their local conformation, due to interactions with nonconserved residues. The chemical shifts of the sequence conserved residues serve to identify which parental rubredoxin presents a local environment for each residue which is most similar to that for the analogous residue in each of the hybrid proteins. The absolute differences in the amide chemical shifts observed between each hybrid and each parental rubredoxin, as well as between the parental proteins themselves, are given in Figure 1. An Ala 2 to Lys variant of Pf rubredoxin (Pf A2K) was utilized as the hyperthermophile reference, due to its efficient N-terminal processing during Escherichia coli expression 23 and its increased similarity with the N-terminal interactions of the Cp protein. In every case for which the amide ¹H chemical shift of a residue in a given hybrid is more similar to that of one parent, the corresponding shift for the other hybrid is more similar to that of the other parent, indicating a clear complementarity in the differential chemical shifts of the two hybrid rubredoxins. Interchange of the two segments containing residues 7 to 11 and 39 to 50 results in the minimum differential chemical shift behavior for both the amide ¹H and ¹⁵N resonances.

Based on this minimum differential amide chemical shift criterion, when all of the mainchain and sidechain ¹H resonances of each hybrid rubredoxin residue are assigned to the corresponding parent, an excellent correlation is observed (Figure 2). The rmsd values for the differential ¹H chemical shifts of the sequence-conserved residues between each of the two metal binding site-swapped hybrids and its corresponding parental rubredoxin are 0.043 and 0.042 ppm (Table 1). In contrast, when these same resonances are compared to the "opposite" parental rubredoxin or compared between the parental Pf and Cp rubredoxins, 4-fold larger rmsd values are obtained. These larger rmsd values are similar to those obtained from the correlation between pairs of homologous proteins having the same level of sequence identity (0.328 ppm for H^N, 0.164 ppm for H^α and 0.246 ppm for H^β at a sequence identity level of 59%) 24.

The close correlation between the chemical shifts of the sequence-conserved residues of the rubredoxin hybrids and the corresponding parental protein chemical shifts indicates that this analysis is sensitive to variations in local conformation which are subtle, relative to the structural differences between the parental proteins. The correlation between the parental and hybrid chemical shifts strongly supports the validity of the proposed structural partitioning based on the differential amide chemical shift data.

The striking correlation in the ¹H shifts illustrated in Figure 2 indicates that, with one exception, the sidechain resonances for every residue in each hybrid protein closely mimic the corresponding sidechain resonances of the parental rubredoxin that is assigned to that hybrid protein on the basis of the backbone amide shifts. Hence, in this protein system, the parsing of cleavage boundaries in forming the "cut-and-paste" models can be effectively done at the residue level, rather than requiring the partitioning of atoms within an individual residue. Only the chemical shifts of Lys 46 H^ε deviate substantially between the hybrids and corresponding parental rubredoxins, as defined by the amide shift comparison. The hybrid-parent chemical shift values for Lys 46 H^ε are displaced symmetrically away from the diagonal in panels A and B of Figure 2, indicating that structural correlation with the "opposite" parent is more appropriate for these protons. The structural significance of this reversed chemical shift correlation for the Lys 46 sidechain will be considered in more detail below.

A differential NOE analysis can utilize either peak heights or peak volumes. NOE volumes are generally used, since they are nominally proportional to the interproton cross relaxation rates, which in turn depend upon the interproton distances. The corresponding peak heights exhibit additional dependencies on local structure and conformational dynamics. However, peak height measurements generally yield higher signal-to-noise values than do peak volumes when a given resonance is monitored among a series of related spectra, as illustrated in spin relaxation studies. The relative statistical advantage of the peak height measurement becomes more enhanced as the intrinsic sensitivity of the experiment decreases, as in the case of the often weak NOE crosspeaks. An additional practical advantage of peak height-based analysis occurs when quantitative comparisons are made among differing resonances, since peak height estimates are generally less sensitive to the selected footprint of the resonance than are the corresponding volume estimates. If the set of analogous parental and hybrid NOE crosspeaks that define the structure of a local region exhibits statistically equivalent peak heights, this provides strong support for a highly similar structure within that region of the proteins. When such equivalent peak heights are not observed, the structural and dynamical effects that underlie the deviations must be resolved by additional analysis.

Figure 3 illustrates the relative peak volumes (panel A) and peak heights (panel B) of well-resolved crosspeaks for 1045 hybrid-parent comparisons from 3D ¹H-¹⁵N-¹H NOESY spectra. This figure only includes the crosspeaks for which both protons in the metal binding site-swapped hybrids are assigned to the same parental (Cp or Pf A2K) rubredoxin structural type, based on the amide chemical shift analysis of Figure 1. The NOE crosspeaks involving Lys 2 H^N, Thr 28 H^γ1 and Phe 49 H^δ, as well as the crosspeaks from sequential H^α-H^N connectivities in conformationally extended residues are indicated by characteristic symbols to highlight their increased deviations from the diagonal. With the exception of these highlighted crosspeaks, the rmsd for the relative peak heights of the corresponding hybrid and parental NOE peaks is 0.022, while the relative rmsd value for the peak volume comparison is 3-fold higher. An rmsd value of 0.043 is obtained for the analogous comparison of 365 hybrid-parental NOE pairs from 2D ¹⁵N suppressed ¹H-¹H NOESY spectra (panel C).

The spread of the data around the diagonal in Figure 3B is largely independent of the absolute peak height value, consistent with the baseplane noise providing the primary source of experimental uncertainty. Comparison between the peak height of the intraresidue Trp 37 H^δ1-H^ε1 crosspeak, used to normalize the 3D NOE intensities, and the baseplane noise level of the various NOESY spectra indicates that the resultant contribution of baseplane noise to the peak height uncertainty is within a factor of 2 of the dispersion around the diagonal. Hence, the structural differences for the corresponding hybrid and parental interactions that underlie the vast majority of the NOE crosspeaks summarized in this figure do not yield variations in peak height that are significantly above the intrinsic noise level of the data.

The structural discrimination offered by our differential NOE analysis can be estimated from the comparison of crosspeaks between pairs of protons on sequence-conserved residues in the parental Cp and Pf A2K rubredoxins. If the same set of resonances as in Figure 3 are filtered out, an rmsd of 0.053 is obtained when the parental 3D NOE crosspeaks are compared. This dispersion is nearly 2 1/2-fold greater than for the matched hybrid-parental NOE data of Figure 3B. Hence, analogous to the differential chemical shift analysis, this differential NOE comparison provides a level of discrimination that is subtle on the scale of the structural variations between sequence-conserved residues in the two parental rubredoxins.

In Figure 3, the NOE crosspeaks arising from each set of connectivities for the Lys 2 H^N, Thr 28 H^γ1 and Phe 49 H^δ protons deviate from the diagonals by approximately proportional amounts. Due to rapid hydrogen exchange of the Lys 2 amide proton, no NOE crosspeaks are observed for either Pf A2K rubredoxin or the metal binding site-swapped Pf rubredoxin hybrid at pH 6.0 and 23°C. Relaxation-compensated CLEANEX-PM experiments 2526 on Cp rubredoxin and the metal binding site-swapped Cp rubredoxin hybrid indicate hydrogen exchange rates under these conditions of 1.5 and 7.0 s^-1, respectively, providing an explanation of the proportionately smaller heights for each of the 10 Lys 2 H^N NOE peaks of this hybrid, relative to those of the parental Cp rubredoxin. Similarly, the markedly broader resonance of Thr 28 H^γ1 in Cp rubredoxin, relative to the corresponding linewidth in the other three proteins, is consistent with more rapid hydrogen exchange for this hydroxyl proton. The 14 pairs of Thr 28 H^γ1 NOE hybrid-parent peak heights vary accordingly. Under the pH and temperature conditions used, hydroxyl proton resonances are commonly not observed, due to rapid chemical exchange with the bulk water resonance 27.

Each of the 21 crosspeaks involving Phe 49 H^δ is consistent with a more modest linebroadening of this resonance in the metal binding site-swapped Cp hybrid and the parental Cp rubredoxin, relative to the other two proteins. In this case, linebroadening due to chemical exchange dynamics must arise from the interchange of the ring protons between differing chemical shift environments in the μs-ms timeframe, most likely due to flipping around the 2-fold symmetry axis of the phenyl group at a rate near the fast exchange limit.

In an extended backbone conformation, the sequential H^α-H^N protons are within van der Waals contact distance. This class of NOE crosspeaks is known to be particularly sensitive to the conformational dynamics effects that arise from asymmetry between the radial and angular components of the interproton vector fluctuations 28. Such heightened sensitivity to asymmetrical fluctuations may account for the increased variability of the peak heights for these crosspeaks (Figure 3B).

It must be emphasized that the marked similarity in peak height for the vast majority of analogous hybrid and parental rubredoxin NOE crosspeaks does not imply an absence of absolute intensity variations due to internal motion for these atoms. Rather, it indicates that in most cases the NOE-effective conformational dynamics of the parental Cp and Pf A2K rubredoxins are faithfully partitioned into the two complementary hybrid structures. This partitioning of dynamics can be illustrated by the aromatic ring of Tyr 13, for which the phenolic hydroxyl proton has 20 and 18 well-resolved NOE crosspeaks in the Cp and Pf A2K rubredoxins, respectively. However, in contrast to the 31 NOE crosspeaks for the Tyr 13 H^δ and H^ε resonances of Pf A2K rubredoxin, there are only three low intensity NOE peaks for the analogous ring protons of Cp rubredoxin, reflecting resonance broadening due to the ring flip dynamics of this sidechain near room temperature. While the Tyr 13 NOE crosspeaks of the metal binding site-swapped Pf hybrid closely mimic those of Pf A2K rubredoxin, the Tyr 13 ring protons of the metal binding site-swapped Cp hybrid have the same three ring proton NOE crosspeaks as observed in Cp rubredoxin and three additional weak crosspeaks that have a maximal normalized amplitude of only 0.035.

When an NOE crosspeak in the rubredoxin hybrids arises from two protons that lie on opposite faces of the hybridization interface, one proton will be assigned to the Cp parental structure type, while the other proton will be of the Pf rubredoxin structure type. Hence, these cross-interface NOE crosspeaks were not included in the hybrid-parent NOE intensity comparisons of Figure 3. In the initial design of the metal binding site-swapped hybrids, for every predicted cross-interface NOE interaction at least one of the two protons is stereochemically equivalent for each of the parental rubredoxins 12. The case in which both protons are stereochemically equivalent in each parental rubredoxin is illustrated by the crosspeak between Phe 30 H^ζ and Lys 46 H^α. These NOE crosspeaks have normalized peak heights of 0.472 in the parental Pf A2K rubredoxin, 0.483 in the metal binding site-swapped Pf hybrid, 0.388 in Cp rubredoxin and 0.325 in the metal binding site-swapped Cp hybrid. This NOE interaction in the metal binding site-swapped Pf hybrid most closely mimics that of the parental Pf A2K rubredoxin, while this interaction in the other hybrid is more similar to that of the parental Cp protein. This pattern is reverse of that predicted from the residue-based parental type assignments utilizing the amide chemical shift data of Figure 1. Hence, the sidechain interactions of Lys 46 appear to follow the parental type of the protein core (Figure 2) rather than the parental type of the metal binding site residues which determine the chemical shift behavior of the Lys 46 amide resonances.

Each metal binding site-swapped hybrid NOE crosspeak between two structurally conserved protons that spans the hybridization interface can be correlated with the more similar of the two analogous parental NOE crosspeaks, while cross-interfacial NOE peaks between a conserved proton and a nonconserved proton can be compared to the corresponding parental rubredoxin peak. Peak height comparisons for the 79 2D and 163 3D cross-interfacial NOE peaks (Figure 4) exhibit a dispersion similar to that for the hybrid crosspeaks of proton pairs assigned to a single parental type (Figure 3). All of the cross-interfacial NOE interactions that have normalized intensities significantly above 0.5 arise from sequential interactions of the amides at the hybrid segment boundaries with the H^α or H^β of the preceding residue, while the large majority of the less intense crosspeaks arise from long range interactions.

The Cp rubredoxin (1.2 Å resolution) 29 and N-Met form of Pf rubredoxin (1.1 Å resolution) 30 X-ray structures were superimposed with a backbone rmsd of 0.63 Å. The coordinates for residues 1–6, 12–38 and 51–54 from Cp rubredoxin were combined with those of residues 7–11 and 39–50 from the Pf protein, to yield the metal binding site-swapped Cp hybrid. Since three of the four metal-coordinating cysteines are provided by the residue 7–11 and residue 39–50 segments, the position of the metal was derived from the Pf rubredoxin structure. The complementary combination yielded the metal binding site-swapped Pf hybrid. No additional modifications of these hybrid rubredoxin models were performed.

At each severed mainchain C-N linkage, the interatomic distance is within 0.23 Å of the optimal bond length, and the apparent bond angles at those junctions are all within 7° of ideal geometry. The length of the regenerated Cys 6 sulfur-metal bond is within 0.05 Å of the parental value for both hybrids. The four mainchain-mainchain hydrogen bonds that were interchanged during the formation of the hybrid structures are regenerated with good geometry. The 3.4 Å contacts of Lys 46 C^ε with both Phe 30 C^ε and Leu 33 C^β are the only interfacial nonbonded interactions to have distances less than 0.90 times the sum of the van der Waals radii for the metal binding site-swapped Pf A2K rubredoxin model. The Phe 49 H^α-Thr 5 O distance of 2.1 Å is the only such interaction in the complementary hybrid structure. Analogous model constructions were carried out with several other X-ray structures of Cp rubredoxin. The 5RXN 31 Protein Data Bank coordinate file yielded a similar quality of stereochemistry at the interface, while the 1IRO 29, 1FHH 32 and 1FHM 32 coordinate sets each produced an interfacial geometry of substantially lower quality.

Lys 46 is highly conserved throughout the family of rubredoxin sequences. The symmetrical displacement of the Lys 46 H^ε chemical shift values from the diagonals in panels A and B of Figure 2 suggests that the local structural environment at the end of this sidechain in each hybrid does not resemble the environment in the parental rubredoxin that is predicted from the Lys 46 mainchain amide chemical shifts, but rather it resembles the environment in the opposite parent. A similar reversed correlation is observed for several Lys 46 sidechain NOE interactions which span the hybridization interface. Figure 5 illustrates the interaction of this sidechain with the backbone segment from Phe 30 to Asp 35 for the NMR-derived structure of each metal binding site-swapped hybrid. As in the parental rubredoxin structures, the ε-amino group of Lys 46 is hydrogen bonded to the mainchain carbonyl oxygens of residues 30 and 33. In the metal binding site-swapped Cp rubredoxin model, the sidechain carboxylate group of Asp 35 bonds with the ε-amino group of Lys 46, as seen in the various X-ray structures of Cp rubredoxin. In contrast, the Asp 35 sidechain is rotated out toward the solvent in the metal binding site-swapped Pf hybrid model (Figure 5), as occurs in the Pf rubredoxin X-ray structure. Given the otherwise highly similar environment of the backbone amide of Asp 35 among the structures, this carboxylate reorientation likely accounts for the nearly 0.4 ppm differential shift of the Asp 35 H^N resonance (Figure 1). In considering what interactions could give rise to this altered conformation, we note that the van der Waals contact of residue 33 (Ile in Cp and Leu in Pf) is the only direct interaction of Lys 46 with a residue that differs between the Cp and Pf sequences.

The metal binding site-swapped Cp rubredoxin crystallized in a P2₁ space group with three protein molecules per asymmetric unit, as earlier observed for a V44A variant of Cp rubredoxin 33. In contrast, the Cp rubredoxin structure 29 used in the NMR-derived structure had crystallized in the R3 space group, with a single molecule in the asymmetric unit. Molecular replacement with the V44A Cp rubredoxin coordinates was used for initial phasing. Refinement was extended to higher resolution with inclusion of heavy atom anisotropic temperature factors and all hydrogen atoms, to attain a final R (R_free) factor of 11.2% (12.5%) for all reflections out to 0.79 Å (Table 2). The rmsd values for the backbones of the A, B and C monomers with respect to the original search model were only 0.22 Å, 0.24 Å and 0.27 Å, respectively.

Figure 6 illustrates the superposition of the three independent molecules of the asymmetric unit along with the NMR-derived structure (red). Molecules A (green) and B (blue) are appreciably more similar to one another than is either of these structures to Molecule C (black). Molecule C diverges most notably from the other three molecules at residues Pro 45, Lys 46 and Ser 47, for which all backbone and sidechain heavy atoms differ by at least 0.5 Å from the superposition with any of the other molecules. Note that the direct interaction between the Lys 46 and Asp 35 sidechains that was predicted in the NMR analysis is indeed observed in the crystal structure.

The average deviations for all sidechain and mainchain heavy atoms in the NMR and X-ray models are given above the diagonal in Table 3 for residues 1 through 52. The analogous calculation for residues 1 through 49 yields a decrease of ~15% in average deviation for the NMR structure, relative to Molecules A (0.317 Å) and B (0.282 Å) in the X-ray structure. The only heavy atoms in the residues 1–49 for which the NMR-derived structure differs from both Molecules A and B by more than 0.5 Å, and furthermore differs from Molecules A and B by more than they differ from each other are: Glu 16 O^ε2, Ile 41 C^δ, Ser 47 O^γ, the carboxyl group of Glu 48 and the C^δ, C^ε and N^ζ atoms of Lys 2 and Lys 46. The sidechains of both Ile 41 and Glu 48 exhibit dual conformers in the electron density map.

A clear difference between the NMR and X-ray structures occurs at the Glu 50 sidechain, for which the χ₁ dihedral angle is 175° in the NMR-derived structure but near +60° in the X-ray structures, despite the fact that the 4.9 Å charge interaction with the Lys 7 sidechain is preserved in both cases. For the metal binding site-swapped Cp hybrid and the parental Pf A2K rubredoxin, the intraresidue NOE crosspeak values between H^α and the degenerate H^β resonances of Glu 50 deviates further from the diagonal than any other nonhighlighted NOE in Figure 3C at (0.379, 0.606). The stronger NOE crosspeak for the hybrid protein is consistent with a switch to a +60° χ₁ angle, for which both β protons are gauche to the H^α. However, the degeneracy of the Glu 50 H^β resonances precluded a clear demonstration of such a conformational shift on the basis of these NOE data alone.

The rubredoxin knuckle structure is observed in nearly all members of the zinc finger superfamily 34. The metal binding site-swapped Cp rubredoxin analysis at 0.79 Å represents the highest resolution Zn²⁺-form protein structure reported to date. Given the marked similarity in Zn²⁺ and Fe²⁺ binding site geometries observed for Cp rubredoxin 2932, the present structure may offer a useful model for the reduced active site as well. Regarding crystal lattice interaction analysis, hydrophobin HFBII 35 is currently the only protein in the Protein Data Bank with multiple nonequivalent monomers in the asymmetric unit which has been reported at a resolution exceeding that of this metal binding site-swapped Cp rubredoxin structure.

The metal binding site-swapped Pf A2K rubredoxin crystallized at pH 5.5 in a P2₁ space group, with eight molecules per asymmetric unit. In contrast, the P2₁2₁2₁ crystals used to determine the Pf rubredoxin structure 30 formed at pH 8.5, with a single molecule in the asymmetric unit. As summarized in Table 2, the refinement for all reflections out to 1.04 Å, utilizing anisotropic B-factors and all hydrogens, yielded a final R (R_free) factor of 13.9% (18.0%).

The average deviations for all sidechain and mainchain heavy atoms of residues 1–52 in the first four molecules of the asymmetric unit are listed below the diagonal in Table 3. A similar set of values is obtained when molecules E through H are compared among themselves. However, when molecules of the first set (A-D) are compared against those of the second set (E-H), the resultant deviations are approximately 1 Å. The asymmetric unit is arranged as four pairs of dimers. Residues 20–23 from molecules A-D form an asymmetric interaction with the corresponding loop residues of their dimer partners of molecules E-H. All of the first four molecules of the asymmetric unit agree with one another to within less than 0.3 Å for all sidechain and mainchain heavy atoms, when residues 20–26 are excluded. It should be noted that the truncated rubredoxin from Desulfovibrio desulfuricans 36 demonstrates that residues 20 to 26 can be deleted without the induction of substantial changes in the rest of the protein structure. Despite the differences in sequence, space group and pH conditions, the backbone coordinates of molecule A differ from those of the original search model by an rmsd value of only 0.22 Å, with modestly larger values for Molecules B, C and D. In contrast, the deviations from the backbone coordinates of the search model were approximately 0.6 Å larger for molecules E-H.

For Molecules A to D of the metal binding site-swapped Pf A2K rubredoxin, sidechains exhibiting dual conformations are Ser 25, Val 44 and Gln 48. The interactions of the three charged residues Lys 29, Glu 31 and Glu 32 vary substantially among the molecules of the asymmetric unit, with the Lys 29 sidechain exhibiting weak electron density. Relative to the X-ray structure, the NMR-derived structure adopts a differing χ₁ dihedral angle for Asp 47 and χ₂ for Glu 50. If 18 sidechain atoms from these five charged residues and Lys 3 are removed from the analysis (out of 401 atoms), the average deviation for all sidechain and mainchain heavy atoms between the NMR-derived structure and Molecule A of the X-ray structure decreases to less than 0.3 Å. The Asp 35 sidechain of the metal binding site-swapped Pf A2K rubredoxin X-ray structure is rotated out toward the solvent phase, away from the Lys 46 N^ζ atom, consistent with the findings of the NMR analysis.

Zn²⁺-form ¹⁵N-labeled samples of Cp rubredoxin, Pf A2K rubredoxin and the complementary metal binding site-swapped hybrids were prepared as previously described 1323. The 2–3 mM protein samples were serially dialyzed against a buffer containing 100 mM sodium chloride, 20 mM sodium phosphate and 20 mM boric acid at pH 6.00. Data collection was carried out on a Bruker DRX 500 at 23°C. 2D COSY and 2D ¹⁵N-suppressed ¹H-¹H NOESY and TOCSY spectra were collected with Watergate suppression 59. 2D ¹H-{¹⁵N}-¹H TOCSY and 3D ¹H-¹⁵N-¹H NOESY spectra were obtained utilizing FHSQC detection 60. Mix times of 200 ms and 83 ms were used for the NOESY and TOCSY spectra, respectively. The NMR data were processed with the FELIX software package (Accelerys, San Diego, CA). NOESY crosspeak heights and volumes were determined using automated peak selection, followed by manual editing in NMRView 61. Relaxation-compensated CLEANEX-PM measurements 2526 on these samples were conducted as previously described 62.

The analysis utilized crosspeaks between pairs of protons separated by at least one flexible dihedral angle. All retained crosspeaks exhibited no significant overlap or other visible spectral distortion and lay within 0.015 ppm (7.5 Hz) and 0.2 ppm (10 Hz) of the assigned chemical shift in the ¹H dimensions and in the ¹⁵N dimension, respectively. The Cp and N-Met Pf rubredoxin X-ray structures were used to exclude all crosspeaks that could potentially arise from more than one pair of protons that are separated by less than 7.5 Å for NOE crosspeaks between amide and methyl protons and less than 6 Å in all other cases. The well resolved 3D NOE crosspeaks analyzed in this study include 83% and 87%, respectively, of all proton pairs predicted to lie within 5 Å of each other in the NMR-derived structures of the metal binding swapped hybrids of the Cp and Pf A2K rubredoxins. Over half of the remainder of predicted crosspeaks corresponds to proton pairs that are separated by at least 4.5 Å. The completeness of the set of analyzed crosspeaks from the 2D NOE spectra is significantly less, due to the much higher fraction of visibly overlapped resonances.

The 3D NOE intensities of the Pf A2K spectrum were normalized to the peak height of the Trp 37 H^δ1-H^ε1 crosspeak. The 3D NOE crosspeak intensities from the Cp and the hybrid rubredoxins were then correlated with the analogous Pf A2K intensities, and the regression line was scaled to unity, so as to adjust for differences in sample concentration and data acquisition reproducibility. The analogous sorting and scaling procedure was applied to the ¹⁵N-suppressed 2D NOE intensities, with the Pf A2K Trp 37 H^ε3-H^ζ3 crosspeak used as internal intensity reference.

The coordinates for Cp rubredoxin [PDB ID:1IRN] 29 and N-Met Pf rubredoxin [PDB ID:1BQ8] 30 were superimposed with LSQMAN 63, using a set of 341 structurally conserved 12 heavy atoms. Hydrogen atoms were added with the program Reduce 64. Atoms for residues 7–11, 39–50, and the metal were interchanged between the Cp and N-Met Pf coordinate sets to generate models of the complementary metal binding site-swapped hybrids. The Lys 2 sidechain of the metal binding site-swapped Pf A2K hybrid was not modeled. Bond lengths and angles were analyzed with Procheck 18, and nonbonded contacts were calculated with Macromodel 65.

Zn²⁺-form protein samples, equilibrated at pH 6.0, were dialyzed against deionized water and then concentrated to 20 mg/ml for the metal binding site-swapped Pf A2K rubredoxin and to 40 mg/ml for the metal binding site-swapped Cp rubredoxin. Crystals were grown at room temperature in hanging drops, by mixing 2 μl of protein solution with an equal volume of reservoir solution. The metal binding site-swapped Cp rubredoxin was crystallized in a solution containing 48% saturated ammonium sulfate, 3% ethylene glycol and 0.1 M sodium acetate at pH 4.5, while the metal binding site-swapped Pf A2K rubredoxin was crystallized in a reservoir solution containing 62% saturated ammonium sulfate and 0.1 M sodium citrate at pH 5.5.

The metal binding site-swapped Cp rubredoxin crystals (0.3 * 0.3 * 0.4 mm) were transferred to a reservoir solution containing 25% glycerol, flash-cooled under a nitrogen stream at 100K and then stored in liquid nitrogen. Prior to flash cooling, the metal binding site-swapped Pf A2K rubredoxin crystals (0.6 * 0.6 * 0.2 mm) were transferred through a series of reservoir solutions with successive 5% increases in ammonium sulfate, to a final concentration of 85% saturated ammonium sulfate to enhance stability during low temperature data collection. To obtain ultrahigh resolution on the synchrotron X-ray beamline X25 of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory, collection was carried out at a wavelength of 0.8577 Å on a ADSC Q315 CCD detector with an oscillation angle and range of 1° and 160°, respectively. Two sets of diffraction data were collected for each crystal, with a different exposure time for each set: 1 sec (to 1.2 Å) and 10 sec (to 0.79 Å) for the metal binding site-swapped Cp rubredoxin crystal, and 3 sec (to 1.4 Å) and 15 sec (to 1.04 Å) for the metal binding site-swapped Pf A2K rubredoxin crystal. All of the data were processed and scaled using HKL2000 66. Although the R_merge value for the highest shell of the metal binding site-swapped Pf A2K rubredoxin was elevated, the nearly 20,000 reflections in this shell were retained due to their good redundancy (3.2) and a high average (I/σI) (2.6).

The metal binding site-swapped Cp rubredoxin crystal is isomorphous with that of the Cp rubredoxin V44A mutant [PDB ID:1C09] 33. Accordingly, the latter structure was used directly for the initial phasing. The structure of the metal binding site-swapped Pf A2K rubredoxin was solved using the PHASER program67, with the 1.1 Å crystal structure of Met-terminal Pf rubredoxin [PDB ID:1BQ8] 30 as the search model.

Structural refinement for both hybrid rubredoxin crystals was initially carried out at 2.0 Å resolution using CNS v 1.0 68, with 5% of the data set aside as the test data for the R_free cross-validation. After initial rigid-body refinement, iterative cycles of positional and temperature factor refinement were carried out, interspersed with model rebuilding into σ_A-weighted (F_o-F_c) and (2F_o-F_c) electron density maps using the program TURBO FRODO 69. At later stages of the structural refinements, the program suite SHELX97 70 was used, with inclusion of all reflection data out to 1.04 Å for the metal binding site-swapped Pf A2K rubredoxin and out to 0.79 Å for the metal binding site-swapped Cp rubredoxin. Each round of refinement consisted of 10–20 cycles of conjugate-gradient least-squares refinement. Water molecules automatically introduced by SHELXH were evaluated manually after each round of refinement. The disorder of main-chain and side-chain atoms was modeled. Anisotropic atomic displacement parameters for all non-H atoms were introduced, resulting in a significant decrease of R_free. After modeling of solvent disorder, the H atoms were added and refined with isotropic temperature factors set by default to 1.2 or 1.5 times the B factor of the bound atom. The Cp and Pf A2K rubredoxin hybrids exhibited dihedral angle deviations of 26.5° and 26.3°, respectively, while the corresponding improper torsion angle values were 1.8° and 1.6°. Atomic coordinates have been deposited in the Protein Data Bank as entries 2PVE (Cp rubredoxin hybrid) and 2PVX (Pf A2K rubredoxin hybrid).