Conformational stability of neuroglobin helix F – possible effects on the folding pathway within the globin family Luca Codutti 1, *, Paola Picotti 2, * , , Oriano Marin 2 , Sylvia Dewilde 3 , Federico Fogolari 1 , Alessandra Corazza 1 , Paolo Viglino 1 , Luc Moens 3 , Gennaro Esposito 1 and Angelo Fontana 2 1 Department of Biomedical Sciences and Technologies and MATI Centre of Excellence, University of Udine, Italy 2 CRIBI Biotechnology Centre, University of Padua, Italy 3 Department of Biochemistry, University of Antwerp, Belgium Introduction Globins are well-known proteins that share the charac- teristic of a typical prosthetic group, traditionally named heme, and corresponding to a protoporphyrin scaffold carrying a single iron ion, formally in the +2 or +3 oxidation state. The metal ion can coordinate several ligands other than protein groups or porphyrin ring atoms. Among the exogenous ligands, molecular oxygen has a specific relevance for the function of Keywords circular dichroism; globin folding; myoglobin; neuroglobin; NMR Correspondence G. Esposito, Dipartimento di Scienze e Tecnologie Biomediche, University of Udine, P. le Kolbe 4, 33100 Udine, Italy Fax: +39 0432 494301 Tel: +39 0432 494321 E-mail: rino.esposito@uniud.it A. Fontana, CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy Fax: +39 049 8276159 Tel: +39 049 8276156 E-mail: angelo.fontana@unipd.it †Present address Institute of Molecular Systems Biology, ETH Zurich, Switzerland *These authors contributed equally to this work (Received 15 April 2009, revised 17 June 2009, accepted 15 July 2009) doi:10.1111/j.1742-4658.2009.07214.x Neuroglobin is a recently discovered member of the globin family, mainly observed in neurons and retina. Despite the low sequence identity (less than 20% over the whole sequence for the human proteins), the general fold of neuroglobin closely resembles that of myoglobin. The latter is a paradigmatic protein for folding studies, whereas much less is known about the neuroglobin folding pathway. In this work, we show how the structural features of helix F in neuroglobin and myoglobin could represent a pivotal difference in their folding pathways. Former studies widely documented that myoglobin lacks helix F in the apo form. In this study, limited prote- olysis experiments on aponeuroglobin showed that helix F does not undergo proteolytic cleavage, suggesting that, also in the apo form, this helix maintains a rigid and structured conformation. To understand better the structural properties of helices F in the two proteins, we analyzed pep- tides encompassing helix F of neuroglobin and myoglobin in the wild-type and mutant forms. NMR and CD experiments revealed a helical conforma- tion for neuroglobin helix F peptide, at both pH 7 and pH 2, absent in the myoglobin peptide. In particular, NMR data suggest a secondary structure stabilization effect caused by hydrophobic interactions involving Tyr88, Leu89 and Leu92. Molecular dynamics simulations performed on the apo and holo forms of the two proteins reveal the persistence of helix F in neu- roglobin even in the absence of heme. Conversely myoglobin shows a higher mobility of the N-terminus of helix F on heme removal, which leads to the loss of secondary structure. Abbreviations Fmoc, 9-fluorenylmethoxycarbonyl; Mb, myoglobin; MbF-P88A, fragment 79–97 of sperm-whale myoglobin with Pro88 replaced by Ala88; MbF-wt, fragment 79–97 of sperm-whale myoglobin; Ngb, neuroglobin; NgbF-A90P, fragment 79–100 of human neuroglobin with Ala90 replaced by Pro90; NgbF-wt, fragment 79–100 of human neuroglobin; NOESY, nuclear Overhauser enhancement spectroscopy; PME, particle mesh Ewald; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy. FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5177 globins, such as myoglobins (Mbs) and hemoglobins, which are generally considered to be oxygen storage and transport proteins [1,2], although other views have been proposed [3,4]. In addition to Mb and hemoglo- bin, over recent years two additional globins have been found to occur in a wide variety of vertebrates, namely neuroglobin (Ngb) [5] and cytoglobin [6]. These two globins exhibit the interesting feature of endogenous hexacoordination of iron [7], whatever its oxidation state. In brief, the metal ion coordinates the four pyr- role nitrogens of the porphyrin group and two imidaz- ole nitrogens of two different histidine residues of the protein, whereas, in Mbs and nearly all hemoglobins, only one iron coordination site is given by a histidine imidazole. The two histidines involved in iron coordi- nation are commonly referred to as proximal and dis- tal, depending on the relative separation from the heme metal, and occur at definite locations of the Mb structural domain. The latter consists of eight helices (A–H) with intervening loops packed with a character- istic fold of two triple-helix layers with nearly orthogo- nal relative rotation (three-over-three) (Fig. 1). The heme group accommodates between the two parallel helices E and F, the proximal histidine being provided by helix F (residue F8) and the distal histidine being provided by helix E (residue E7), according to consen- sus numbering [7]. Although the distal histidine bind- ing to the metal ion is the signature of endogenous hexacoordination of Ngbs and cytoglobins, only the proximal histidine coordination occurs invariably in all globins. ApoMb, the heme-free Mb, retains the highly helical fold of native Mb at neutral pH. However, NMR [8,9] and limited proteolysis [10,11] studies have shown that helix F in apoMb is disordered and readily cleaved by proteases. The limited proteolysis pattern also led to the establishment that no other potential cleavage sites of apoMb undergo hydrolysis, as a consequence of the stability of the helical fold of the protein [11]. An important determinant responsible for the conforma- tional flexibility of the chain segment encompassing helix F in apoMb has been recognized in the nature of residue F3, a proline residue that disrupts the local a-helical conformation and destabilizes significantly the whole helix F (Fig. 1). Indeed, substitution of the helix- breaking Pro88 (F3) residue with the helix-forming ala- nine residue in sperm-whale apoMb successfully meets expectations and apparently restores the local helix geometry, as inferred from CD profiles and limited proteolysis [11], although none of the techniques can distinguish partial from full restoration. In these earlier studies [8,11], it was proposed that helix F in the native holoprotein is stabilized by interactions with the heme moiety, counterbalancing the helix-breaking effect of proline. As a proline residue at location F3 occurs in more than 90% of Mb sequences and several hemoglo- bin chains, these globin species in their apo form should exhibit low, if any, helical propensity in the corresponding helix F segment. This is in agreement with the proposed main folding pathway of apoMb at neutral pH, sketched as U fi AGH fi ABGH fi ABCDEGH fi N, where U and N are the unfolded A B C Fig. 1. Three-dimensional structure of human Ngb (A) and sperm-whale Mb (B). The models were constructed from the X-ray structure of the Ngb mutant C46G ⁄ C55S ⁄ C120S (PDB code 1OJ6, chain B) and sperm-whale Mb (PDB code 1VXD). Helix F is highlighted by a white ellipsoid in both diagrams. (C) Sequences of the pep- tides that were addressed in the present study, i.e. helix F encompassing fragments 79–100 of Ngb and 79–97 of sperm-whale Mb, together with the corresponding vari- ants. A box highlights the actual extension of helix F in the parent protein structures. The wild-type sequences are indicated as NgbF-wt and MbF-wt; the variant sequences are identified by the correspond- ing mutations, i.e. NgbF-A90P and MbF- P88A, respectively. Globin helix F conformational stability L. Codutti et al. 5178 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS and native conformations, respectively, and A, B, etc. are the helix segments [12]. The folding scheme confirms the absence of a stably folded helix F in native apoMb, in contrast with the N state of the holoprotein [12]. The stability of the globin domain has been addressed recently by comparing the apo forms of horse Mb and human Ngb at acidic pH (P. Picotti, unpublished results). The well-established instability of apoMb at low pH [13,14] was confirmed. At variance with the extensive loss of apoMb secondary structure, the apoNgb chain was observed to preserve most of the helical fold at acidic pH and limited proteolysis experiments suggested that only the N-terminal frag- ments were sufficiently flexible to become susceptible to proteolytic cleavage. Therefore, among the pre- served helical segments of apoNgb, there was also helix F. This finding appears to be consistent with the absence of a proline residue in the chain segment encompassing helix F in apoNgb (Fig. 1). A useful approach to the study of the mechanism of protein folding entails the analysis of the confor- mational preferences of isolated peptide fragments. Short linear peptide fragments cannot exhibit the tertiary interactions that they establish in the intact proteins. Therefore, the assessed conformational trends of isolated fragments are the same as those occurring in the protein chain during the early stages of folding, when only local and inherent conforma- tional propensities drive the folding process. Along these lines, a direct CD and NMR investigation was carried out previously in order to establish the folding propensities of Mb peptide fragments [15]. In addition to confirming inherent helix propensities for AB and GH segments, this study also showed that the helix F fragment has quite a low propensity towards helical geometry, even in the presence of 2,2,2-trifluoro- ethanol (TFE). In order to investigate more deeply the suggested differences in helix F stability within the globin family, limited proteolysis of apoNgb at neutral pH and the intrinsic conformational stability of peptides encom- passing the different helix F variants (Fig. 1) are addressed here. Indeed, it is shown that helix F in apo- Ngb has a strong propensity for a-helical secondary structure, at variance from helix F in apoMb. These results allowed us to infer that the folding pathway of apoNgb is different from that of apoMb, despite the similarity of their overall fold. The conclusion reached in this study reinforces the view that the same protein structural topology does not imply the same folding pathway. An analogous view was also expressed in a comparative study of the folding pathway of Mb [12] and leghemoglobin [16]. Results and discussion Limited proteolysis Limited proteolysis experiments were performed on wild-type human apoNgb at neutral pH with the enzyme thermolysin (Fig. 2). Compared with the incu- bation times typically required by horse or sperm- whale apoMb, i.e. seconds [10,11], apoNgb proteolysis proved to be much slower. After 4 min of incubation, apoNgb shows only two sites of preferential cleavage, i.e. at the level of the N-terminal helix (helix A), pre- cisely between Ala15 and Val16, and at the interhelical segment between helix F and helix G, precisely between Ala98 and Val99. The proteolysis pattern of the latter region also involves hydrolysis between Ser91 and Leu92, with the formation of fragment 16–91, which becomes the predominant species at longer protease incubation times. The later onset of fragment 16–91 demonstrates that it derives from further proteo- lytic digestion of the initially formed species 16–98 at the level of the newly exposed C-terminus. However, a Fig. 2. Limited proteolysis of human apoNgb at neutral pH. Proteol- ysis of apoNgb by thermolysin (enzyme to substrate ratio 1 : 100 by weight) was conducted at 25 °Cin50m M Tris-HCl, 0.15 M NaCl, pH 7.0. The proteolysis mixture was analyzed by reverse-phase HPLC after 4 and 30 min of incubation. The identities of the protein fragments were established by electrospray mass ionization mass spectrometry and are indicated by the labels near the chromato- graphic peaks. L. Codutti et al. Globin helix F conformational stability FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5179 significant amount of undigested full-length protein is still present at either proteolysis intervals. Therefore, the cleavage location is partially dependent on the incubation time of the substrate with the protease, at least within the limits of the experimental protocol. Cleavage at the level of the turn-like fragment, joining helix F to the rest of the C-terminal region, suggests that this region is highly flexible in Ngb at neutral pH, and thus it is a proper protease substrate. The addi- tional cleavage site at the N-terminal region of Ngb (at the end of helix A) recalls the previously observed limited proteolysis pattern of apoMb at acidic pH, with a cleavage extended to most of helix B at low pH [13]. Strikingly, at both pH conditions, helix F of human apoNgb does not undergo proteolytic cleavage as instead observed in apoMbs, under either neutral [10,11] or acidic conditions [13], suggesting that the apoNgb helix F maintains a sufficiently rigid structure to prevent proteolysis. These results are perfectly in line with the agadir [17] secondary structure predic- tions reported in Fig. 3, which illustrates the helical propensity for the whole Ngb sequence, at neutral and acidic pH, and for wild-type or mutated helix F peptides of Ngb and Mb. CD analysis Far-UV CD measurements (Fig. 4) of the peptides under investigation were conducted in different experi- mental conditions in order to analyze their content of secondary structure. The investigated peptides encom- pass the native sequences of helix F in sperm-whale Mb (MbF-wt) and human Ngb (NgbF-wt) and the corresponding mutants obtained by the replacement of Pro88 (ProF3, in the globin consensus map [7]) with an alanine in Mb (MbF-P88A), and of Ala90 (AlaF2) with a proline in Ngb (NgbF-A90P) (see Fig. 1). Fig- ures 4A,B depict the spectra obtained for NgbF-wt at neutral pH conditions with increasing amounts of TFE and in aqueous solution at decreasing pH, respectively. At neutral pH, the CD spectrum of NgbF-wt displays two prominent minima at 208 and 222 nm, typical of a-helical polypeptides. The helix content of NgbF-wt steadily increases with TFE, from 35% without organic solvent to 56% at 20% TFE. On lowering the pH (Fig. 4B), instead, the helix content decreases from the same initial value as in Fig. 4A to 30% at pH 2.2. The mutation of Ala90 into proline destroys the helix content of the parent sequence, as evident from the corresponding CD spectrum typical of random coil peptides (Fig. 4C). The addition of TFE restores some helix content (17%) in NgbF-A90P, to an extent, how- ever, much below that observed for NgbF-wt. Far-UV CD spectra collected on the peptides encompassing the sequence of helix F in Mb are reported in Fig. 4D. As expected from previous results on the whole protein and mutants thereof [11], as well as on isolated frag- ments [15], the peptide MbF-wt, with the natural sequence bearing a proline in position F3, displays very little helical content at neutral pH, whereas the peptide MbF-P88A, where the proline is replaced by alanine, exhibits a slightly higher helix content (16%). It is worth noting that the experimental helix content obtained from CD data for the isolated peptides paral- lels the expectations obtained using agadir semi- empirical predictions on the corresponding native and mutant full-length proteins (Fig. 3, right). NMR analysis 1 H NMR spectra were collected only for the NgbF-wt fragment at two different pH values, i.e. pH 6.3 and Ngb, pH 2.0 Ngb, pH 7.0 Ngb A90P Mb WT Mb P88A Ngb WT Fig. 3. Left: helical propensity of the poly- peptide sequences of Ngb at neutral and acidic pH calculated using the AGADIR algo- rithm [17]. The locations of the eight helices (A–H) along the polypeptide chain of the protein are also indicated by boxes, accord- ing to the structural features obtained from the PDB record. Right: AGADIR-predicted heli- cal propensities for helix F of wild-type human Ngb, A90P human Ngb, wild-type sperm-whale Mb and P88A sperm-whale Mb. Globin helix F conformational stability L. Codutti et al. 5180 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS pH 2.1 in water, and pH 6.3 in 10% aqueous TFE. Detailed analysis was performed, however, only for the datasets obtained in water to which we will refer, unless otherwise indicated. Spin systems were first identified, for both pH conditions, in the total correla- tion spectra, and then assigned on the basis of nuclear Overhauser enhancement spectroscopy (NOESY) map sequential connectivity patterns [18]. A general over- view of the NMR information shows that, for both series of experiments, no long-range restraints (interac- tions between nuclei more than five residues apart) were detected. Figure 5 displays the distribution along the sequence of all collected restraints under both experimental conditions. Secondary structure meaning- ful cross-peaks have been found for both pH condi- tions and are shown in Fig. 6. Even a cursory examination suggests that typical helix conformational patterns occur in the central region of the investigated peptide at both pH conditions, whereas the N-terminal and C-terminal segments appear to be poorly struc- tured. An additional interesting feature that emerges from nuclear Overhauser enhancement restraints is the occurrence, at both pH conditions, of medium-range hydrophobic interactions between Tyr88 H d or H e and Leu85, Leu89, Leu92 H d . After removing all redundancies, the experimental restraint sets consisted of 211 meaningful interatomic distances for experiments made at pH 6.3, and 236 meaningful interatomic distances for experiments made at pH 2.1. The two series formed the experimental databases for the subsequent restrained modeling. Table 1 summarizes the final output of restrained modeling. Structural validation performed using the software aqua and procheck-nmr [19] confirmed the presence of a regular a-helix secondary structure at both pH conditions, with slightly different lengths. At pH 6.3, the a-helix involves residues from Glu86 to Leu92, whereas, at pH 2.1, the a-helix extends from Glu87 to Ser91, in qualitative agreement with the estimates 10 AB CD NgbF, pH 7.2 –10 0 0% TFE 10% TFE –20 20% TFE [θ] × 10 –3 (deg·cm 2 ·dmol –1 ) Wavelength (nm) 200 210 220 230 240 250 0 NgbF –10 –5 pH 2.2 pH 7.2 pH 4.1 –15 [θ] × 10 –3 (deg·cm 2 ·dmol –1 ) Wavelength (nm) 200 210 220 230 240 250 NgbF, pH 7.2 –5 0 –15 –10 NgbF-A90P NgbF-A90P, 20% TFE [θ] × 10 –3 (deg·cm 2 ·dmol –1 ) Wavelen g th (nm) 200 210 220 230 240 250 MbF, pH 7.2 MbF-P88A [θ] × 10 –3 (deg·cm 2 ·dmol –1 ) –5 0 –20 –15 –10 MbF Wavelen g th (nm) 200 210 220 230 240 250 Fig. 4. CD characterization of peptides encompassing helix F of human Ngb and sperm-whale Mb (see Fig. 1). (A) Far-UV CD spectra of NgbF-wt peptide dissolved in 50 m M Tris-HCl ⁄ 0.15 M NaCl, pH 7.0, in the presence of different amounts of TFE. (B) Far-UV CD spectra of NgbF-wt peptide dis- solved in 10 m M HCl, pH 2.2 or pH 4.1. The spectrum at pH 7.2 is redrawn for compari- son. (C) Far-UV CD spectra of NgbF-A90P peptide dissolved in 50 m M Tris-HCl ⁄ 0.15 M NaCl, pH 7.0, in the presence of 20% TFE. (D) Far-UV CD spectra of MbF-wt and MbF-P88A peptides dissolved in 50 m M Tris-HCl ⁄ 0.15 M NaCl, pH 7.0. All spectra were recorded at 25 °C. L. Codutti et al. Globin helix F conformational stability FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5181 obtained from CD data. Over these fragments, the average upfield deviations of H a chemical shifts from the values of statistically disordered structures [20] are 0.21 ± 0.11 and 0.19 ± 0.13 p.p.m. under neutral and acidic conditions, respectively (the corresponding value in TFE is 0.25 ± 0.12 p.p.m.). Such deviation extents are above the chemical shift index threshold to validate helical tracts [21], which, for a linear peptide in water, suggests that a helical geometry is locally significantly populated. Validation of the secondary structure for the remaining residues in both pH conditions con- firmed a statistically disordered state. According to procheck-nmr, at both pH conditions, refined struc- tures showed no dihedral angle of the fragment 86–92 in disallowed Ramachandran regions. The helical seg- ments also revealed low accessibility because of a Number of constraints Number of constraints Residue number Residue number A B Fig. 5. Restraint distribution along the sequence of the NgbF-wt peptide. The restraints obtained at pH 6.3 (A) and pH 2.1 (B) and subsequently used for simulated annealing calculations are given. In the histograms, white represents intraresidue restraints, light grey sequential restraints and dark grey medium-range restraints. Fig. 6. Secondary structure diagnostic restraints obtained at pH 6.3 (A) and pH 2.1 (B). The bar thickness is proportional to the corre- sponding nuclear Overhauser enhancement intensity. Table 1. CYANA 2.1 and DISCOVER output parameters for NgbF-wt restrained molecular dynamics calculations and subsequent refinement. Structure family at pH 6.3 Average Range CYANA Average backbone rmsd to mean ⁄ 10 )1 nm 0.56 ± 0.28 0.33–1.39 Average heavy atom rmsd to mean ⁄ 10 )1 nm 0.89 ± 0.28 0.57–1.59 Target function ⁄ 10 )2 nm 2 (6.23 · 10 )2 )± (1.85 · 10 )2 ) Violated distance constraints 0 Violated van der Waals’ constraints 0 DISCOVER Average backbone rmsd to mean ⁄ 10 )1 nm 0.37 ± 0.21 0.07–1.03 Average heavy atom rmsd to mean ⁄ 10 )1 nm 1.33 ± 0.32 0.68–2.45 Structure family at pH 2.1 Average Range CYANA Average backbone rmsd to mean ⁄ 10 )1 nm 0.36 ± 0.13 0.27–0.65 Average heavy atom rmsd to mean ⁄ 10 )1 nm 0.59 ± 0.15 0.43–0.94 Target function ⁄ 10 )2 nm 2 (0.28 ± 4.53) · 10 )2 Violated distance constraints 0 Violated van der Waals’ constraints 0 DISCOVER Average backbone rmsd to mean ⁄ 10 )1 nm 0.26 ± 0.13 0.04–0.56 Average heavy atom rmsd to mean ⁄ 10 )1 nm 1.06 ± 0.38 0.14–1.78 Globin helix F conformational stability L. Codutti et al. 5182 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS back-fold trend of the disordered flanking regions (Fig. 7). Additional validation parameters are reported in Table S1 (see Supporting information). A fitting of the 20 final conformers (at both pH con- ditions) over the a-helix validated zone led to mean backbone rmsd deviation values of 0.037 ± 0.021 nm for structures calculated at pH 6.3 and 0.026 ± 0.013 nm for structures calculated at pH 2.1. Additional details are given in Table 1. Figure 8 shows a diagrammatic representation of the superposition of the structure of family conformers at both pH conditions. The structures were superimposed to minimize rmsd within the regular helical fragments in both cases. As apparent from the side-chain distri- bution, it is likely that hydrophobic interactions between Tyr88 and both Leu89 and Leu92 side-chains create a scaffold capable of stabilizing the local helical fold in either pH conditions. Indeed, the chemical shifts of the Leu92 side-chain isopropyl moiety are shifted upfield by 0.13–0.18 p.p.m., whereas Leu89 H b resonances occur downfield with respect to the basic aqueous shift value [20] by 0.18–0.20 p.p.m. at both pH conditions. Molecular dynamics simulations Snapshots have been taken at 100 ps intervals in order to obtain a statistical ensemble for the three systems studied. We consider first the molecular dynamics sim- ulations of the apo forms of Mb and Ngb in order to check whether any difference in dynamics could be highlighted even in a simulation time as short as 3 ns. Although the loss of secondary structure for apoMb is expected, it is not obvious how fast this process may be. Molecular dynamics simulation shows that the N-terminal part of helix F (entailing residues Glu83 to Leu86) loses its helical conformation in the first 200 ps of simulation. In particular, the u and w angles formed by these residues are quite different from those of stan- dard a-helices and exhibit very large fluctuations. There is no clear conformational transition towards completely different conformations, but overall the backbone is very flexible. The results concerning helix F are in agreement with earlier simulation studies [22,23]. This picture is further confirmed by the analy- A B Fig. 7. Overlay of the 20-membered conformer families of the NgbF-wt peptide. Superpositions were obtained by fitting the struc- tured regions observed at different pH conditions: (A) pH 6.3; (B) pH 2.1. A B Fig. 8. A diagrammatic view of the structured regions of the 20 peptide family members at both pH conditions: (A) pH 6.3; (B) pH 2.1. The side-chains involved in the a-helical secondary structure are highlighted in red. L. Codutti et al. Globin helix F conformational stability FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5183 sis of backbone rmsds, which are larger than 0.2 nm at residues 83 and 84. In contrast, apoNgb maintains a standard a-helical conformation at the corresponding residues (Glu86 to Leu89). The a-helix does, however, start for most of the time at residue Ser83. The hypothesis put forward in this work is that Tyr88 plays a crucial role in scaffolding residues 85, 89 and 92. Indeed, when these residues are superimposed, the average heavy atom rmsd between any two pairs of snapshots is 0.088 ± 0.026 nm, therefore showing a rather stable arrange- ment of these amino acids. This rmsd value can be compared with that obtained for the corresponding residues in apoMb, which is 0.126 ± 0.042 nm. In order to check that the loss of ordered conformation in apoMb was not a simulation artifact, a 3 ns molecular dynamics simulation was performed on the holo form of the same protein. In this simulation, the helical con- formation is preserved, as expected during all simula- tions, because of the additional constraint on the helix provided by the covalently bonded heme group. In order to further validate the NMR results, 1.2 ns molecular dynamics simulations were run on the stud- ied peptides. The results further confirmed all the available evidence. For the MbF-wt fragment, the heli- cal conformation is lost after 500 ps at the N-terminal residues and the helix is found mostly between residues Leu86 and Ala94. The missing backbone amide proton of proline seems to be the determinant of secondary structure loss at the N-terminus, according to the typical CO(i)–HN(i + 3), CO(i)–HN(i + 4) hydrogen- bonding pattern of helical conformations. In contrast, for the NgbF-wt peptide, the regular helical conforma- tion is maintained after 500 ps, from Ser83 to Val99. Interactions among hydrophobic moieties of Leu85, Leu89, Tyr88 and Leu92 appear to be particularly rele- vant in conferring stability to the helix. Ngb helix F The collection and interpretation of structural data at neutral and acidic pH conditions highlight the interest- ing features of human Ngb helix F structuring. Previ- ous evidence has shown that helix F of apoNgb is preserved from proteolysis at pH 2 (P. Picotti, unpub- lished results), suggesting the conservation of its sec- ondary structure in spite of the extreme conditions. By contrast, at the same pH value, apoMb underwent extensive proteolysis [13], whereas, at neutral pH, proteolytic cleavage occurred only at helix F [10,11]. In the present work, a clear persistence of the a-helical structure in strong acidic conditions has also been con- firmed for the isolated NgbF-wt peptide. A first glance at the amino acid charge position over this peptide sequence led us to postulate initially a secondary struc- ture stabilization as a result of favorable charge inter- action with the a-helix macrodipole [24,25] of Glu86 and Glu87 side-chains. Hence, decreasing the pH to a value of 2.1 should have affected the whole helix stability because of a loss of the side-chain-mediated electric shielding from Glu86 and Glu87 carboxylates. As mentioned previously, a decrease in pH decreases the a-helix extension, from residues 86–92 to 87–91, but does not totally disrupt it. This means that, in the addressed sequence, the main helix-nucleating driving forces are likely to arise from other structuring energy contributions. One such contribution may arise from the interactions that could be established in NgbF-wt between Glu86 and Glu87 amides and the carbonyl and side-chain oxydryl acceptors of the preceding serine pair, in particular Ser84 occurring in the ideal position for N-capping [26] (no similar N-capping potentiality is present in MbF-wt). However, our exper- imental evidence does not support this N-capping occurrence in NgbF-wt, but rather hydrophobic inter- actions. Medium-range interactions revealed by NMR NOESY spectra at both pH conditions involve princi- pally Tyr88, Leu89 and Leu92, arranged in a helical geometry with an ideally suited separation between Tyr88 and Leu92. This experimental evidence is compatible with the hydrophobic scaffold-mediated hypothesis advanced above. The limited proteolysis pattern observed for apoNgb after 30 min of incubation with thermolysin is in line with this interpretation. Indeed, the proteolytic cleav- age affecting helix F, which is unprotected as a result of the loss of segment 99–151, occurs between residues Ser91 and Leu92, i.e. at the C-end of the proposed hydrophobic scaffold, despite the fact that an even more favorable thermolysin proteolytic site can be rec- ognized between Tyr88 and Leu89. Interestingly, equivalent results were also observed when thermolysin hydrolysis was performed on the isolated NgbF-wt peptide (Fig. S3, see Supporting information). Indeed, by aligning the known Ngb sequences obtained from the UniProtKB ⁄ Swiss-Prot database (http://www. expasy.org/cgi-bin/get-similar?name=globin%20family), a general motif can be recognized to occur in all F-helices: {L 82 -[SH]-[ST]-L-E-[ED]-[YF]-L-X-X-L-G- [R,K]-K-H-[R,Q]-A 98 }. In addition to the invariant His96 (proximal HisF8), which is expected because of its essential role in heme coordination, Leu92 (LeuF4) is also well conserved in the globin family [27] and, indeed, it has structural relevance in maintaining the position of the His96 imidazole ring [7]. In addition, the Ngb subfamily is specifically characterized by the Globin helix F conformational stability L. Codutti et al. 5184 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS occurrence of a conserved aromatic residue at position 88 (tyrosine or phenylalanine). The recognized motif also presents phylogenetic persistence of negatively and positively charged (at physiological pH) clusters close to the N- and C-termini of the helix, respectively. Finally, the presence of conserved leucines, three to four residues apart, is most noticeable, an arrangement that creates, with the mentioned aromatic residue at position 88, the hydrophobic face of an amphipathic helix (see Fig. 9). The regularly spaced leucine residues are likely to contribute to the extension of the helix F N-terminal side via hydrophobic stacking. This is con- sistent with the NMR evidence obtained for NgbF-wt in aqueous TFE (10%), which suggests some propen- sity to helix elongation, namely to a helix also involv- ing the N-terminal fragment 79–85 (Figs. S1 and S2, see Supporting information), in agreement with the conspicuous helix content increase also observed by CD under similar conditions. Inspection of the crystal structure of human Ngb reveals the relevance of the helix F amphipathicity. Although the heme surface contacts the upper side of the helix F hydrophobic face, a crucial contact involves Leu89, i.e. the first leu- cine of the helix F hydrophobic scaffold, and Met144 of helix H (Cc 89 –Cc 144 = 0.441 nm). As Met144 is invariant within Ngb sequences, it can be proposed that the positioning of the helix F hydrophobic scaf- fold may be dictated by helix H, i.e. a strongly persis- tent secondary structure element that has always been recognized to be involved in the early folding events, at least in Mb [12] and leghemoglobin [16]. Conclusions The inherent conformational properties of isolated protein fragments have often been used to analyse pro- tein folding pathways [28]. As fragments cannot develop the long-range interactions of native proteins that usually form along the folding pathway of the whole protein chain, the propensity of a protein frag- ment to adopt a precise secondary structure appears to be relevant to the early protein folding events. The results of this study indicate that a peptide encompass- ing helix F of Ngb has a strong propensity to adopt an a-helical secondary structure in water solution, as given by far-UV CD and NMR measurements. As the isolated Ngb helix F autonomously forms a hydropho- bic helical scaffold, we advance the hypothesis that hydrophobic interactions within the core segment 88–92 of Ngb helix F could represent the primary helix-forming driving force that contributes to the ini- tial helix core during the folding of apoNgb. In partic- ular, the presence of a conserved tyrosine residue at position 88 appears to provide a very stable arrange- ment of nearby residue side-chains in helix F, thus making the helical geometry quite stable. In addition, the hydrophobic scaffold 88–92 appears to be suitably located to establish a favourable hydrophobic contact with a conserved residue of helix H, probably an early- folding one by analogy with previous evidence [12,16]. Although helix F formation seems to occur early in the folding pathway of Ngb, all models of apoMb folding pathways so far developed do not include the structuring of helix F [16,23,29–31]. Molecular dynam- ics simulations also support this difference. Therefore, we suggest a different folding pathway for Ngb and Mb, with helix F being an early nucleating folding core in Ngb, rather than the last folding step as in Mb, where helix F is formed only on addition of the heme moiety. The globin family has been used previously as an excellent experimental system for analysing protein folding mechanisms, as the helical globin fold is highly conserved between proteins with widely differing amino acid sequences [32]. It has been proposed that the folding pathways of evolutionarily related proteins with similar three-dimensional structure, but different sequences, should be similar [33,34]. Mb and Ngb share an almost superimposable three-dimensional fold and show a low degree of sequence identity (less than 20% over the whole sequence for the human proteins). In this study, we conclude instead that, despite the Fig. 9. A ball-and-stick view of the crystallographic structure of human Ngb helix F with a heme ring. Hydrogen bonds are high- lighted in green with the corresponding distances calculated by Swiss PDB viewer. L. Codutti et al. Globin helix F conformational stability FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5185 strong similarity of the overall fold of Mb and Ngb, these two proteins display different folding pathways. A similar scenario has emerged already from the com- parison of Mb and leghemoglobin, two proteins with the same type of folded structure, but adopting differ- ent folding pathways [16]. These two proteins form rapidly compact helical folding intermediates that direct the overall folding pathway of the whole poly- peptide chain, but the details of the pathways are dif- ferent and depend on the local amino acid sequences. Although apoMb forms an A(B)GH helical intermedi- ate [29], leghemoglobin initially forms an intermediate given by helices G and H and part of helix E [16]. Moreover, recently, it has been shown that the molecu- lar details of the intermediate formed by leghemoglo- bin in kinetic experiments differ from those of the equilibrium molten globule intermediate [35]. There- fore, individual proteins, such as Ngb, Mb or leghemo- globin, despite their overall fold similarity, can follow different folding pathways dictated by the solution conditions and differences in amino acid sequences [36]. Experimental procedures Materials Thermolysin from Bacillus thermoproteolyticus was pur- chased from Sigma (St. Louis, MO, USA). Solvents, resin and coupling reagents for peptide synthesis were obtained from Applied Biosystems (Foster City, CA, USA). All pro- tected amino acids were purchased from Novabiochem (Laufelfingen, Switzerland). HPLC-grade solvents were obtained from Merck (Darmstadt, Germany). The expression and purification of the Ngb mutant C120S was performed as described previously [37]. The Cys120 to serine replacement in Ngb was made in order to avoid protein aggregation processes of the apo form of the protein (apoNgb) as a result of the formation of an inter- molecular disulfide bond. The preparation of apoNgb was obtained from the corresponding holoprotein by the removal of heme by reverse-phase HPLC separation. Briefly, the holoprotein was loaded onto a C 18 Vydac col- umn (4.6 · 250 mm; The Separations Group, Oak Ridge, TN, USA), eluted with a linear gradient of water–acetoni- trile, both containing 0.05% (v⁄ v) trifluoroacetic acid (TFA), from 5 to 40% in 5 min and from 40 to 60% in 25 min, at a flow rate of 0.8 mLÆmin )1 . The effluent was monitored by absorption measurements at 226 nm and fractions containing the protein were pooled and then con- centrated in a SpeedVac system. The possible contamina- tion of the apoprotein preparation by the holoprotein was assessed spectrophotometrically, and no significant absorp- tion was observed in the Soret region. Peptide synthesis The peptides used in this study were designed to reproduce chain segments 79–100 of human Ngb and 79–97 of sperm- whale Mb and were produced as N-acetylated and C-ami- dated species. In addition to the wild-type peptides, two variants were also studied bearing a single residue replace- ment. The amino acid sequences of the peptides used herein are shown in Fig. 1B. The peptides were synthesized by solid-phase peptide synthesis using an automated peptide synthesizer (model 431-A; Applied Biosystems). The 9-flu- orenylmethoxycarbonyl (Fmoc) strategy was used through- out the peptide chain assembly [38]. As solid support the 4-(2¢,4¢-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyace- tamido-norleucylaminomethyl resin (Rink amide AM resin) (Novabiochem) (loading of 0.74 mmmolÆg )1 ) was used. The side-chain-protected amino acids used were as follows: Fmoc-Asp(tert-butyl), Fmoc-Glu(tert-butyl), Fmoc-Ser (tert-butyl), Fmoc-Thr(tert-butyl), Fmoc-Tyr(tert-butyl), Fmoc-Gln(trityl), Fmoc-His(trityl), Fmoc-Lys(tert-butyloxy- carbonyl) and Fmoc-Arg(2,2,4,6,7-pentamethyldihydroben zofuran-5-sulfonyl). Coupling was performed with a single reaction for 45 min by a 0.45 m solution in N,N¢-dimethyl- formamide of 2-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluro- nium hexafluorophosphate and N-hydroxybenzotriazole in the presence of N-ethyldiisopropylamine, following the man- ufacturer’s protocols. At the end of the solid-phase synthe- sis, the peptidyl-resins were acetylated by treatment with 10% acetic anhydride in N,N¢-dimethylformamide to yield an N-acetylated peptide. Cleavage of the crude peptides was performed by reacting the acetylated peptidyl-resins with TFA–H 2 O–thioanisole–eth anedithiol–p henol ( 10 mL : 0 .5 mL : 0.5 mL : 0.250 mL : 750 mg) for 2.5 h. The peptides were pre- cipitated with ice-cold ethyl ether and isolated by centrifuga- tion. The pellets were washed several times with ether, dissolved in water and lyophilized. Crude peptides were purified by a preparative reverse-phase HPLC column (PrepNova-Pak HR C 18 , 250 mm · 10 cm, 6 lm bead size; Waters, Milford, MA, USA) at 12 mLÆmin )1 using a linear gradient of 5–50% acetonitrile in 0.08% TFA. The molecu- lar masses of the peptides were confirmed by electrospray mass ionization mass spectrometry using a Micro Q-Tof mass spectrometer (Waters, Manchester, UK). The purities of the purified peptides were 98% as evaluated by analytical reverse-phase HPLC. Proteolysis experiments Limited proteolysis experiments with thermolysin were con- ducted on apoNgb at 25 °C with the proteins dissolved (0.5 mgÆmL )1 )in50mm Tris-HCl, 0.1 m NaCl, 1 mm CaCl 2 , pH 7.0, using an enzyme to substrate ratio of 1 : 100 (by weight). At time intervals, aliquots were taken from the reaction mixture and proteolysis was stopped by the acidification of the solutions by adding TFA (final con- Globin helix F conformational stability L. Codutti et al. 5186 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... Nature of the charged-group effect on the stability of the C-peptide helix Proc Natl Acad Sci USA 82, 234 9–2 353 26 Aurora R & Rose GD (1998) Helix capping Protein Sci 7, 2 1–3 8 27 Ota M, Isogai Y & Nishikawa K (1997) Structural requirement of highly-conserved residues in globins FEBS Lett 415, 12 9–1 33 Globin helix F conformational stability 28 Wright PE, Dyson HJ & Lerner RA (1988) Conformation of peptide... peptide fragments of proteins in aqueous solution: implications for initiation of protein folding Biochemistry 27, 716 7–7 175 29 Eliezer D, Yao J, Dyson HJ & Wright PE (1998) Structural and dynamic characterization of partially folded states of apomyoglobin and implications for protein folding Nat Struct Biol 5, 14 8–1 55 30 Jamin M & Baldwin RL (1996) Refolding and unfolding kinetics of the equilibrium folding. .. intermediate of apomyoglobin Nat Struct Biol 3, 61 3–6 18 31 Jamin M (2005) The folding process of apomyoglobin Protein Pept Lett 12, 22 9–2 34 32 Bashford D, Chothia C & Lesk AM (1987) Determinants of a protein fold: unique features of the globin amino acid sequences J Mol Biol 196, 19 9–2 16 33 Krebs H, Schmid FX & Jaenicke R (1983) Folding of homologous proteins: the refolding of different ribonucleases... independent of sequence variations, proline content and glycosylation J Mol Biol 169, 61 9–6 35 34 Stackhouse TM, Onuffer JJ, Matthews CR, Ahmed SA & Miles EW (1988) Folding of homologous proteins: conservation of the folding mechanism of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium and five interspecies hybrids Biochemistry 27, 82 4–8 32 35 Nishimura C, Dyson HJ &... (c) the conformation of chain A in the crystal structure of Ngb (PDB code 1OJ6) with the heme group removed Protons were added using the pdb2gmx utility of the gromacs simulation package [52] The protonated structure was then used to generate a topology and coordinate file using the psfgen utility of namd simulation software [53] The structure was used to compute the electrostatic potential around the. .. Casadio R & Irace G (2000) The effect of tryptophanyl substitution on folding and structure of myoglobin Eur J Biochem 267, 393 7–3 945 23 Onufriev A, Case DA & Bashford D (2003) Structural details, pathways, and energetics of unfolding apomyoglobin J Mol Biol 325, 55 5–5 67 24 Blagdon DE & Goodman M (1975) Letter: mechanisms of protein and polypeptide helix initiation Biopolymers 14, 24 1–2 45 25 Shoemaker KR,... acid-destabilized forms Biochemistry 40, 512 7–5 136 15 Reymond MT, Merutka G, Dyson HJ & Wright PE (1997) Folding propensities of peptide fragments of myoglobin Protein Sci 6, 70 6–7 16 16 Nishimura C, Prytulla S, Dyson HJ & Wright PE (2000) Conservation of folding in evolutionary distant globin sequences Nat Struct Biol 7, 67 9–6 86 17 Munoz V & Serrano L (1997) Development of the multiple sequence approximation within. .. centration, 0.1%) The proteolysis mixtures were then separated by reverse-phase HPLC utilizing a C18 Vydac column in the experimental conditions described previously The identity of the fragments was established by electrospray mass ionization mass spectrometry using a Micro Q-Tof mass spectrometer (Waters) CD spectroscopy Globin helix F conformational stability analysis were performed using felix software... compilation ª 2009 FEBS L Codutti et al 13 Picotti P, De Franceschi G, Frare E, Spolaore B, Zambonin M, Chiti F, Polverino de Laureto P & Fontana A (2007) Amyloid fibril formation and disaggregation of fragment 1–2 9 of apomyoglobin: insight into the effect of pH on protein fibrillogenesis J Mol Biol 367, 123 7–1 245 14 Gilmanshin M, Gulotta M, Dyer RB & Callender RH (2001) Structures of apomyoglobin’s various... by 300 conjugate gradient steps using periodic boundary conditions and the particle mesh Ewald (PME) method for electrostatic interactions [56] PME employed a grid of 128 · 128 · 128 points The PME tolerance was set to 10)5 nm)1 which, together with the cut-off of 1.2 nm, resulted in a Ewald coefficient of 2.57952 nm)1 The minimized system was further relaxed, keeping the solute (including the ion) fixed, . Conformational stability of neuroglobin helix F – possible effects on the folding pathway within the globin family Luca Codutti 1, *, Paola Picotti 2, * , ,. variance from helix F in apoMb. These results allowed us to infer that the folding pathway of apoNgb is different from that of apoMb, despite the similarity of their overall fold. The conclusion reached in. conditions. Inspection of the crystal structure of human Ngb reveals the relevance of the helix F amphipathicity. Although the heme surface contacts the upper side of the helix F hydrophobic face,