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. 2005 Dec;14(12):3057–3063. doi: 10.1110/ps.051742605

Modulation of oxygen binding to insect hemoglobins: The structure of hemoglobin from the botfly Gasterophilus intestinalis

Alessandra Pesce 1, Marco Nardini 2, Sylvia Dewilde 3, David Hoogewijs 4, Paolo Ascenzi 5, Luc Moens 3, Martino Bolognesi 2
PMCID: PMC2253232  PMID: 16260762

Abstract

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 Å crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).

Keywords: Gasterophilus intestinalis hemoglobin, parasitic botfly hemoglobin, insect hemoglobin, heme hexa-/penta-coordination, oxygen recognition, protein structure

Hemoglobin (Hb) and related heme proteins reversibly bind gaseous diatomic ligands (e.g. O2, CO, and NO) as the sixth axial ligand of a penta-coordinate heme, in their deoxygenated form (Antonini and Brunori 1971; Bolognesi et al. 1997). However, several members of the Hb superfamily have been reported to display a functionally relevant hexa-coordinate heme Fe atom, in their deoxygenated state, where the fifth axial ligand is the invariant proximal HisF8 residue, and the sixth axial ligand is generally the heme distal HisE7 (Hargrove et al. 2000; Pesce et al. 2002; de Sanctis et al. 2004a; Weber and Fago 2004). Binding of exogenous ligands to hexa-coordinate globins is thus a complex event, characterized by (1) removal of the endogenous sixth ligand bound to the heme Fe atom, (2) formation of a transient reactive heme penta-coordinate species, and (3) binding of the exogenous ligand (e.g., O2) to the vacant heme Fe sixth coordination site (Duff et al. 1997). Because of competition of diatomic exogenous ligands with the endogenous HisE7 residue for coordination to the heme, the high intrinsic affinity displayed by O2, CO, and NO for the transient reactive penta-coordinate heme species is significantly reduced, yielding apparent affinities similar to those typical of naturally occurring penta-coordinate globins (e.g., sperm whale myoglobin, Mb) (Bolognesi et al. 1997; Hargrove et al. 2000; Pesce et al. 2003, 2004; Burmester and Hankeln 2004; de Sanctis et al. 2004b, 2005; Hoy et al. 2004; Vallone et al. 2004a,b; Weber and Fago 2004; Weiland et al. 2004; Brunori et al. 2005; Hankeln et al. 2005). In addition to the above mechanistic considerations, HisE7 may play a gating role modulating access/exit of exogenous ligands to/from the heme distal pocket (“E7 gate”), and may stabilize the heme-bound ligand through hydrogen bonding, thus achieving a pivotal role in the regulation of ligand affinities (Bolognesi et al. 1982, 1997; Perutz 1989; Springer et al. 1994).

Respiration in adult insects has generally been considered to be adequately supported by their efficient tracheal system, connecting inner organs to the air; no respiratory proteins have therefore been deemed as necessary to support body O2 diffusion (Brusca and Brusca 1990). Nevertheless, recent data locate intracellular Hbs in a large variety of insects, indicating that O2 supply in insects can be more complex than previously thought, and may partly rely on Hbs facilitating O2 uptake, transport, and storage (Burmester and Hankeln 1999; Hankeln et al. 2002, 2005; de Sanctis et al. 2005).

Thus far, insect Hbs belonging to three different families have been characterized. Chironimids exhibit a stage-specific and tissue-specific synthesis of Hbs throughout larval and pupal stages. Twelve different globin chains, and more than 30 globin genes, have been identified in Chironomus thummi thummi (Goodman et al. 1983; Green et al. 1998; Bergstrom 1999), a similar situation being described for the Japanese midge species Tokunagayusurika akamusi (Yamamoto et al. 2003). Extracellular Chironomus thummi thummi HbIII (CttHbIII) (Fig. 1 ▶) matches the classical three-on-three α-helical globin fold, and displays a penta-coordinate heme in the deoxygenated form, with the HisE7 residue in an “open gate” conformation (Steigemann and Weber 1979). An intracellular Hb (DmHb), expressed in both the larvae and the adult insect, has been described in the fruit fly Drosophila melanogaster (Fig. 1 ▶). Within the classical globin fold, DmHb hosts a bis-His hexa-coordinate heme Fe atom in the deoxygenated derivative, the axial ligands being HisF8 and HisE7 (Burmester and Hankeln 1999; Hankeln et al. 2002; de Sanctis et al. 2005). The larvae of the parasite botfly Gasterophilus intestinalis, living attached to the inside of horse stomach, host several Hb isoforms at millimolar concentration in their highly tracheated posterior spiracular plate cells. G. intestinalis Hbs enable the larvae to make efficient use of intermittent contact with the air swallowed with food (Keilin 1944; Phelps et al. 1972; Wittenberg 1992). Despite the high sequence homology to DmHb (37% residue identity) (Fig. 1 ▶), G. intestinalis Hb1 (GiHb1) hosts a penta-coordinate heme Fe atom in the deoxygenated form (Dewilde et al. 1998). Although different structural behaviors of the heme distal site in insect Hbs may underlie distinct O2 recognition, binding, and stabilization mechanisms, CttHbIII, DmHb, and GiHb1 show comparable O2 affinities, close to that of sperm whale Mb (P50 values of 0.12–0.46 torr; see Table 1) (Springer et al. 1994; Dewilde et al. 1998; Hankeln et al. 2002).

Figure 1.

Figure 1.

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Amino acid sequence alignment of insect Hbs compared to the reference sequence of sperm whale Mb. The α-helices of the classical globin fold are labeled A through H above the alignments. Highly conserved residues (ProC2, PheCD1, HisE7, and HisF8) are highlighted in gray. SwMb, sperm whale Mb; GiHb1, Gasterophilus intestinalis Hb1; DmHb, Drosophila melanogaster Hb; CttHbIII, Chironomus thummi thummi HbIII.

Table 1.

O2 binding to insect Hbs and sperm whale Mb

Ligands
O2
kon ×10−7 (M−1 s−1) koff (s−1) P50 (torr) P50*a (torr) HisE7 Ka Heme Fe atom HisE7 conformation
C. t. thummi HbIIIb 30 218 0.46 — — Penta-coordinate Open gate
D. melanogaster Hbc 6.4 1.0 0.12 0.0063 18 Hexa-coordinate —
G. intestinalis Hb1d 1.0 2.4 0.15 — — Penta-coordinate Closed gate
Sperm whale Mbe 1.9 10 0.33 — — Penta-coordinate Closed gate
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aP50* = P50/(1 + Ka) (Hankeln et al. 2002).

b From Burmester et al. 2002.

c From Hankeln et al. 2002.

d From Dewilde et al. 1998.

e From Antonini and Brunori 1971.

In the context of our ongoing studies aiming at shedding light on the growing complexity of O2 recognition and stabilization mechanisms in hexa- and penta-coordinate Hbs (de Sanctis et al. 2004a,b, 2005; Pesce et al. 2004), we report here the crystal structure of oxygenated GiHb1, isolated from larvae, at 2.6 Å resolution.

Results and Discussion

The three-dimensional structure of oxygenated GiHb1 was solved by molecular replacement methods, using the DmHb structure (de Sanctis et al. 2005) as search model, and refined at 2.6 Å resolution (R-factor = 18.6%, R-free = 25.5%, with ideal stereochemical parameters) (Engh and Huber 1991). The final model contains 2614 protein atoms (for two independent GiHb1 chains), six ordered solvent atoms, and two O2 molecules (see Table 2). The RMSD between the two asymmetric unit chains is 0.3 Å, calculated on 149 Cα pairs. The largest deviations occur in the CD-D region (0.9 Å at Asp(52)D3) and at the C terminus, resulting from crystal contacts.

Table 2.

Data collection and refinement statistics

Data collection
    Resolution range (Å) 27.0–2.6
    Independent reflections 11,160
    I/σ(I) 7
    R-merge (%) 9.1
    Completeness (%) 98
Refinement
    Refinement resolution range (Å) 27.0–2.6
    Reflections used in refinement 10,033
    No. of protein atoms 2614
    Water molecules 6
    Overall B-factor (Å2) 45
    RMSD from ideal values:
        Bond lengths (Å) 0.016
        Bond angles (°) 0.7
    Ramachandran plota
        Residues in most favored regions 92.1%
        Residues in additional allowed regions 7.9%
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a Data produced using the program PROCHECK (Laskowski et al. 1993).

The presence of a homodimer in the crystallized GiHb1 (the two subunits will be referred to as A and B) had previously been anticipated by solution studies on the dissolved crystals (Dewilde et al. 1998), in agreement with earlier purification results (Keilin and Wang 1946). The GiHb1 asymmetric unit dimer assembly is based on a rather extended association interface (844 Å2), contributed by the G- and H-helices of each chain (Fig. 2A ▶). The four α-helices (G–H and G′–H′; structural elements from subunit B are primed) are related by a local twofold axis running along the association interface, being in contact for most of their extensions and yielding a nearly classic anti-parallel four-α-helix bundle. The dimer association interface is based on the twofold repetition of several polar contacts; in particular, association of the two chains is based on electrostatic interactions (one set of contacts) of the pairs Asp112–His130′, Lys111–Asp137′, and Asn108–Asp141′, and by interactions mediated by a water molecule (w5) involving residues Asp112, Glu116 (from chain A), Lys134′, and His130′ (from chain B). The quaternary assembly achieved by GiHb1 has not been observed before within the globin superfamily, although G- and H-helices individually are exploited in the assembly of different globin oligomers, for example in Vitreoscilla sp. Hb (Bolognesi et al. 1997; Tarricone et al. 1997). As a result of GiHb1 quaternary assembly, the two hemes are far apart (32 Å between the two heme Fe atoms), their propionate groups pointing in opposite directions. Despite dimerization, no cooperativity in ligand binding to GiHb1 has been reported (Dewilde et al. 1998).

Figure 2.

Figure 2.

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Overall views of GiHb1 quaternary and tertiary structures. (A) The Cα trace of both GiHb1 subunits A (cyan) and B (yellow), building up the observed homodimer. For each subunit the heme group is drawn in red, and the dimerization interface α-helices are labeled. Some of the key residues building up salt bridges stabilizing the quaternary association are drawn. (B) Display of the structural overlay of GiHb1 and DmHb. The two proteins (A subunit for GiHb1, cyan ribbon; DmHb, magenta skeleton, PDB entry code 2BK9) are overlaid based on their Cα backbones. The figure shows the location adopted by the two heme groups following overlay of the penta-coordinate GiHb1 on the hexa-coordinate DmHb. The proximal HisF8 and distal HisE7 residues are shown to highlight the different distal site stereochemistry in the two cases (GiHb1 distal site includes the Fe-coordinated O2 molecule, in orange). Helices E and F, distal and proximal to the heme, are labeled. Note the subtle structural shifts that allow heme distal and proximal backbone readjustments supporting hexa- versus penta-coordination. Drawn with MOLSCRIPT (Kraulis 1991).

GiHb1 displays a strongly conserved globin fold (overall sequence identity 21% to sperm whale Mb, RMSD 1.3 Å, calculated over 134 Cα pairs), the largest structural deviation matching a six-residue insertion at the EF interhelical hinge (Fig. 1 ▶). Additionally, GiHb1 is structurally related to DmHb (sequence identity 37%, RMSD 1.3 Å, calculated over 138 Cα pairs) (see Fig. 2B ▶). Moreover, structural overlays with CttHbIII (sequence identity 13%) yield a 1.7 Å RMSD value, limited to 115 Cα pairs, due to slightly different orientations of the α-helical segments in the two Hbs.

The overall surface of each GiHb1 monomer appears highly rich in charged residues also outside the above-mentioned association interface, the total number of negatively and positively charged residues being 25 and 27, respectively. The two Cys residues present in the protein at sites (119)G17 and (121)G19 are 5.4 Å apart (Cα–Cα distance), with their side chains pointing in opposite directions. Inspection of the electron density, and stereochemical considerations, indicate that they are not involved in intramolecular or intermolecular disulphide bridges.

Stabilization of the heme group within the GiHb1 fold occurs through 31 van der Waals contacts (≤4.0 Å); moreover, electrostatic interactions from the distal E-helix to the heme propionates provide additional stabilizing contributions. In particular, two salt links are present between residue Arg(58)E3 and heme D-propionate, and between residue Arg(65)E10 and heme A-propionate. These two Arg residues together with Arg (68)E13 build an evident positively charged patch around the heme crevice, located on the heme distal side.

Concerning the heme axial ligands, the proximal His(97)F8 residue is coordinated to the Fe atom through a 2.0 Å bond (in both the A and B subunits). In the A chain, the O2 molecule is bound to the heme Fe atom via a 1.9 Å coordination bond, adopting a rather bent geometry, the FeO1O2 angle being 100° (a comparable arrangement is observed in the B subunit). The heme-Fe-bound dioxygen molecule is stabilized by a hydrogen bond to His(62)E7 NE2 atom (3.1 Å, and 2.6 Å for subunits A and B, respectively). The heme distal site is essentially composed of apolar residues that contribute an uncommon Phe–Pro–Trp–Phe sequence motif at sites CD1-CD4, creating a hydrophobic bulky cluster next to the conserved Phe(42)CD1 and the heme. Likely related to the overall apolarity, but also to the steric constraints posed by the above-mentioned residues, no water molecules are observed in the heme distal cavity of the crystallized oxygenated GiHb1.

Penta-coordinate insect GiHb1 and mammalian sperm whale Mb host the distal HisE7 residue hydrogen-bonded to the heme-bound O2 (achieving the “closed E7 gate” conformation), whereas CttHbIII displays the HisE7 residue swung in an “open E7 gate” conformation (Steigemann and Weber 1979; Yang and Phillips 1996; Fig. 3 ▶). Nevertheless, GiHb1, sperm whale Mb, and CttHbIII show oxygen affinities comparable to that of DmHb (Table 1), which displays endogenous heme Fe hexa-coordination (de Sanctis et al. 2005). Notably, ligand affinity in DmHb is controlled by rupture of the intramolecular heme Fe-HisE7 bond, the apparent value of P50 for O2 binding (0.12 torr) being ~20-fold higher than that of the intrinsic parameter P50* (0.0063 torr) for the penta-coordinate species (see Table 1). On the other hand, the apparent kinetic parameters for O2 binding/dissociation to/from GiHb1, DmHb, and sperm whale Mb match each other, but differ substantially from those reported for CttHbIII (de)oxygenation (Table 1). Such observations, and consideration of the kinetic parameters of Table 1, suggest that a wide variety of O2 binding mechanisms are operative in insect Hbs, where opening of the “E7 gate” and rupture of the Fe-HisE7 bond may represent the rate-limiting steps for O2 binding to GiHb1 and DmHb, respectively. The high second-order rate constant observed for CttHbIII oxygenation may be related to the “open gate” conformation of the HisE7 residue, fully swung out of the heme distal pocket by the steric repulsion of residue IleE11 (Bolognesi et al. 1997; Fig. 3B ▶). The low first-order rate constant for O2 dissociation from GiHb1, DmHb, and sperm whale Mb reflects the stabilization of the heme Fe bound ligand by hydrogen bonding to the HisE7 residue (Springer et al. 1994; Hankeln et al. 2002). In contrast, fast dissociation of the CttHbIII heme bound O2 reflects lack of stabilization through hydrogen bonding to residues in the heme distal site (Steigemann and Weber 1979).

Figure 3.

Figure 3.

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Stereo views of the closed and open HisE7 gate conformations in GiHb1 and in CttHbIII. (A) Details of the heme distal site structure adopted by GiHb1 in its oxygenated form (present report; the O2 molecule is shown in orange), where the distal HisE7 is in the “closed gate” conformation. (B) The “open gate” conformation of HisE7, swung out of the heme distal pocket, is shown for CttHbIII deoxygenated form (PDB entry code 1ECD; a water molecule, not bound to the heme Fe atom, at the entrance of the distal pocket is shown in magenta). For both panels, the heme group is shown edge on, in red, and part of the heme distal E-helix is shown as a ribbon. Drawn with MOLSCRIPT (Kraulis 1991).

The structural data so far available indicate that transition from hexa- to penta-coordination in globins may be achieved either by shifting of the N-terminal half of the E-helix away from the heme pocket, as observed in human cytoglobin (de Sanctis et al. 2004a), and in DmHb (de Sanctis et al. 2005; D. de Sanctis and M. Bolognesi, unpubl.), or by sliding of the heme within its crevice, as reported for mouse neuroglobin (Vallone et al. 2004b). A structural comparison of GiHb1 and DmHb, based on superposition of their Cα backbones, shows that backbone matching between the two proteins is slightly better on the distal side of the heme (1.3 Å divergence at the Cα atoms of the distal HisE7 residues) rather than on the proximal side, where the F-helices of the two proteins diverge by ~1.7 Å at the Cα atom of residue HisF8. A moderately different tilt and location of the heme group in the two proteins is also observed, resulting in the Fe atom being moved by ~1.1 Å in the direction of the distal HisE7 residue in DmHb relative to GiHb1. Moreover, the heme crevice aperture (as measured by the distance between HisE7 and HisF8 Cα atoms) is smaller by 2 Å in DmHb. Heme shift toward the distal site and reduced room between the heme distal and proximal helices (E and F, respectively), being the result of many distributed structural contributions, are both factors promoting heme bis-His hexa-coordination in DmHb.

Related to the above concepts, it should be noted that heme hexa-coordination has also been reported for more distant globins whose structures are known. For example, nonsymbiotic rice Hb displays a hexa-coordinate heme, based on the HisE7 sixth ligand, and a disordered CD-D region (Hargrove et al. 2000). Synechocystis sp. truncated Hb has been shown to achieve hexa-coordination through binding of HisE10 to the heme Fe atom (Scott and Lecomte 2000; Hoy et al. 2004), thus inducing notable conformational transitions in the heme distal region. Such transitions, however, can hardly be compared to those discussed above, given the modified fold (two-on-two helical sandwich) adopted by Synechocystis sp. truncated Hb.

The crystal structure of GiHb1 adds a new component to the insect Hb family, whose main properties have been analyzed here in a comparison focused on Hb heme hexa- versus penta-coordination. The different structural responses provided by insect globins to what may appear to be a very similar heme cavity event (i.e., O2 coordination to the heme Fe atom) further underline that the achievement of endogenous heme hexa-coordination by a given globin, and the ensuing control of heme reactivity versus exogenous biatomic ligands, is the result of very subtle residue mutations and conformational changes, distributed over wide and different regions of the globin fold, rather than being ascribable to evident and well localized residue mutations. In keeping with these observations, the three-dimensional structures of CttHbIII, DmHb, and GiHb1, as well as the detailed analysis of their ligand binding parameters, show that an overall almost constant affinity for O2 may reflect very different ligand recognition and binding mechanisms, part of which (i.e., heme hexa-coordination) has been thus far underestimated. The functional meaning of such structural and mechanistic variability (even within the same animal species), besides hinting at evolutionary convergence toward average O2 equilibrium binding properties, is an open matter of investigation.

Materials and methods

Protein purification

G. intestinalis live specimens were obtained from a local slaughterhouse. The spiracular plates were dissected, immediately frozen in liquid nitrogen, and powdered. After extraction in 50 mM Tris-HCl (pH 8.1), in the presence of a protease inhibitor cocktail (Complete, Boehringer) and 0.5% (w/w) polyvinylpolypyrrolidone, the suspension was cleared by centrifugation (10 min at 30,000g, followed by 45 min at 110,000g); GiHb1 was precipitated from the supernatant in the 65%–95% ammonium sulphate saturation range (Phelps et al. 1972). Purification through hydrophobic interaction chromatography was performed on a phenyl-Sepharose CL 4B column, equilibrated in 1.8 M ammonium sulphate, 50 mM Tris-HCl (pH 8.1). GiHb1 was eluted using 0.1 M potassium phosphate buffer (pH 7.0) and further purified by ion exchange and gel-filtration chromatography. The final purification step was performed by semipreparative iso-electric focusing (Dewilde et al. 1998).

Crystallization and data collection

GiHb1 crystals were grown via the vapor diffusion technique, at a protein concentration of 30 mg/mL, using a precipitant solution containing PEG 4000 25% (w/v), 20 mM Tris (pH 7.5–8) at 4°C. These crystals (about 0.15 × 0.15 × 0.4 mm3) grew in ~2 wks as rounded hollow hexagonal prisms, as reported (Dewilde et al. 1998). Remarkably, crystals grown from recombinant GiHb1, prepared as described (Dewilde et al. 1998), were of lower diffraction quality; optimization of their growth conditions was not pursued. For data collection at cryotemperature (100 K) the crystals were transferred in the same storage solution, supplemented with 20% (v/v) glycerol. X-ray diffraction data were collected on a Mar345 image plate detector, coupled to a Rigaku RU-200 rotating anode generator (Cu, Kα), to 2.60 Å resolution. Diffracted intensities were processed and reduced to structure factors using Mosflm (Leslie 1992) and programs from the CCP4 suite (CCP4 1994; see Table 2). Inspection of the diffracted intensities showed that GiHb1 crystals belong to the trigonal space group P31 (or P32), with unit cell constants a = b = 47.7 Å, c = 145.2 Å, γ = 120°. Calculation of the crystal packing parameter (VM = 2.72 Å3/Da, 55% solvent content) indicated the presence of two GiHb1 molecules in the asymmetric unit, in agreement with the results of the calculated protein self-rotation function.

Structure determination and refinement

Structure solution was achieved through molecular replacement techniques, using the program MolRep (Vagin and Teplyakov 1997). The crystal structure of hexa-coordinate DmHb was used as search model, considering the heme group and only six α-helices (excluding the C and D helices) trimmed of the connecting loops, with side chains truncated to Ala in cases of mismatch between the two amino acid sequences. The rotational and translational searches (trigonal space group P31), run in the 27–3 Å resolution range, yielded a prominent solution for a dimeric molecule, with a correlation coefficient of 32.1% and a corresponding R-factor of 52.6%.

Initially the two GiHb1 molecules were refined using the program CNS (Brünger et al. 1998), with a run of rigid body refinement, moving independently all of the helices and the heme group in order to avoid bias due to the DmHb search model, and simulated annealing, immediately showing the electron density for the loop and helical regions deleted in the molecular replacement search model. Model building/inspection was based on the program O (Jones et al. 1991). Subsequently, the two complete GiHb1 molecules were refined using the CNS (Brünger et al. 1998) and REFMAC (Murshudov et al. 1997) programs. At the end of the refinement stages, six water molecules were located through the inspection of difference Fourier maps, using the program O. The final R-factor value was 18.6% (for all the reflections in the 27.0–2.6 Å resolution range), and R-free 25.5% (see Table 2). Atomic coordinates and structure factors have been deposited with the Protein Data Bank (Berman et al. 2000), with entry codes 2c0k e r2c0ksf, respectively.

Acknowledgments

We thank Dr. Alessio Bocedi for helpful discussions. This work was supported by grants from the Ministry for University and Scientific Research (project RBAU015B47_002) and the European Union (project QRTL-2001-01548). We are grateful to Dr. Kristina Djinovic-Carugo for help with X-ray data collection at EMBL Heidelberg (Germany). S.D. is a postdoctoral fellow of the FWO (Fund for Scientific Research Flanders). M.B. is grateful to CIMAINA (Milano, Italy), and to Fondazione Compagnia di San Paolo (Torino, Italy) for continuous support.

Abbreviations

  • Hb, hemoglobin

  • Mb, myoglobin

  • RMSD, root mean square deviation

  • CttHbIII, Chironomus thummi thummi HbIII

  • DmHb, Drosophila melanogaster Hb

  • GiHb1, Gasterophilus intestinalis Hb1

  • amino acid residues have been labeled according to their sequence number (in parentheses) and their topological position within the globin fold (Perutz 1989).

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051742605.

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