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Protein Science (2002), 11:2273-2278.
Copyright © 2002 The Protein Society

FOR THE RECORD

Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A 1H-NMR and electronic absorbance study

Daniela Delli Castelli1, Elena Lovera1, Paolo Ascenzi2 and Mauro Fasano3

1 Department of Chemistry "IFM," University of Torino, I-10125 Torino, Italy
2 Department of Biology, University "Roma Tre", I-00146 Rome, Italy
3 Department of Functional and Structural Biology, University of Insubria, I-21100 Varese, Italy

Reprint requests to Mauro Fasano, Department of Structural and Functional Biology, University of Insubria, Via Jean H. Dunant 3, I-21100 Varese, Italy; e-mail: mauro.fasano{at}uninsubria.it; fax: 39-0332-421500.

(RECEIVED April 11, 2002; ACCEPTED June 18, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0210202.


   Abstract
TOP
 Abstract
Introduction
 Materials and methods
 References
 
The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N{longleftrightarrow}X{longleftrightarrow}D, where X represents a disordered globin structure (occurring at ~4 M urea) that still binds the heme.

Keywords: Caretta caretta; nuclear magnetic resonance; sea turtle myoglobin; unfolding; urea


   Introduction
TOP
 Abstract
 Introduction
Materials and methods
 References
 
Myoglobins (Mbs) are monomeric globular proteins devoted to the storage and facilitated diffusion of oxygen in cardiac and striated muscles (see Wittenberg and Wittenberg 1989). Very recently, Mb has been reported to catalyze nitric oxide scavenging, protecting cellular respiration (Brunori 2001). Moreover, Mb has been postulated to protect Trypanosoma cruzi in cardiomyocites from the antiparasitic effect of nitric oxide (Ascenzi et al. 2001).

From the structural viewpoint, Mb belongs to the globin family, a class of all-{alpha} heme binding proteins consisting of six/eight helices (A to H) connected by short random coils. Members of this family share a high conservation of the three-dimensional fold, despite a sequence conservation as low as 16%. The six/eight helix regions dock together to form an hydrophobic core, allocating the prosthetic heme group. The most conserved residues in Mbs are those involved in heme contacts, in fold stabilization, and in helical packing (see Ptitsyn and Ting 1999).

Evolutionarily distant globins have been shown to reach the same final fold through different folding pathways, such as the case of mammalian and leguminosae apoHbs (Nishimura et al. 2000). Actually, much has been understood on folding intermediates of apoproteins, the mildly acidic folding intermediate I of apoMb at pH 4 (i.e., the molten globule) having been the subject of intense scrutiny (Hughson et al. 1990; De Sanctis et al. 1994; Eliezer and Wright 1996; Kay and Baldwin 1996; Staniforth et al. 2000). Nevertheless, a mechanistic view of the folding pathway of native holoMb has not yet been completely defined (Janes et al. 1987; Chiba et al. 1994; Wittung-Stafshede et al. 1998; Moczygemba et al. 2000).

Loggerhead sea turtle (Caretta caretta) Mb is the only reptile hemoprotein with a three-dimensional structure that has been solved. This monomeric Mb shows a tertiary structure that strictly conforms to the classic globin fold, and values of interhelical angles are conserved (Nardini et al. 1995). Although reptiles and mammals diverged 300 Myr ago, C. caretta and sperm whale (Physeter catodon) Mbs share 63% amino acid identity. Nevertheless, some peculiarities should be noticed. The negatively charged residue at position (5)A3 (Asp or Glu) is characteristic of turtles and lizards, whereas mammals have Gly, and birds and alligators have Asn. Moreover, (35)B16 residue is Leu instead of Val or Ala. The characteristic Asp44-Lys47 salt bridge in the CD corner is missing (residue 44 is Ala), and residue (52)D2 is hydrophobic (Ile), thus conferring a peculiar hydrophobicity to this region (Nardini et al. 1995). On the other hand, 10 ion pairs have been observed in the crystal structure, connecting parts of the tertiary structure located quite far apart, including those considered critical for docking of the B helix to the GH {alpha}-{alpha} hairpin (Nardini et al. 1995; Ramos et al. 1999). Most of the hydrophobic residues involved in interhelical contacts are conserved, except for Val13 (Ile in sperm whale), Ile115 (Leu in sperm whale), and Ser144 (Ala in most mammals); the latter substitution is observed in man as well (Herrera and Lehmann 1971). Tryptophanyl residues at positions (7)A5 and (14)A12 are conserved in C. caretta Mb (Nardini et al. 1995).

Here, the effect of urea concentration on the model of the backbone solution structure of the cyanide derivative of ferric C. caretta Mb, at pH 5.4, is reported. Structural data have been obtained by constrained torsion angle dynamics by means of nuclear Overhauser effects (NOEs) measured at different urea concentrations (Cavanagh et al. 1996). Sequential and long-range contacts have been used as conformational parameters and compared with unfolding data obtained by electronic absorption spectroscopy in the Soret region.

The achievement of a three-dimensional structure by nuclear magnetic resonance (NMR) for a protein larger than 8–10 kD usually requires full isotopic labeling with magnetically active 13C and 15N nuclei. In fact, the complete assignment of side-chain resonances and detection of NOEs between the residues is hampered by both crowding of the fingerprint region and excessive linewidth of the observed signals (Cavanagh et al. 1996). The partial assignment of the proton resonances of C. caretta Mb was achieved by means of TOCSY, DQF-COSY, and NOESY experiments (Cavanagh et al. 1996), by taking advantage of the X-ray crystallographic coordinates (Protein Data Bank code 1LHT; Nardini et al. 1995) to help the assignment of interresidue contacts. Accordingly, 322 resonances belonging to 80 residues out of 155 were unambiguously assigned. Although it is far from being representative of the tertiary structure at a reasonable definition, detection of sequential NOE is sufficient to define secondary structure elements, and NOE peaks between nonsequential residues allow us to draw a diagram of the global fold of the protein (Cavanagh et al. 1996; Freund et al. 1996; Ragona et al. 1999). The resulting model shows a good superimposition to the crystallographic structure (Fig. 1AGo), with a root mean square deviation value of 1.6 Å for the backbone atoms of all helical regions.



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  		Fig. 1. (A) Superimposition of the nuclear magnetic resonance (NMR) model (in red) with the X-ray diffraction structure of Caretta caretta myoglobin in blue; Protein Data Bank code 1LHT (Nardini et al. 1995). The root mean square deviation value is 1.6 Å for the backbone atoms of all helical regions. The heme group (from the X-ray structure) is rendered in green. (B) Urea denaturation curves of C. caretta Mb: long-range NOEs (filled circles), sequential NOEs (filled squares), and Soret band absorption (open diamonds). Best-fit parameters according to Equation 1Go are reported in Table 1Go. (C) Unfolding of the ABCD helices at different urea concentrations. Blue indicates without urea; green, 1.5 M; yellow, 2.0 M; and red, 2.2 M. (D) Unfolding of the EFGH helices at different urea concentrations. Blue indicates 1.5 M; green, 2.2 M; and orange, 3.0 M. The blue structure shows no appreciable differences with that without urea.

 
By addition of urea as the chaotropic denaturant, NOE peaks are more or less gradually lost, thus permitting us to follow changes either in the global fold motif or in its secondary structure elements. Figure 1BGo reports the folded fraction (Ff; see Equation 1Go) as obtained from the relative number of observed NOESY cross peaks as a function of the denaturant concentration for short- and medium-range contacts, as well as for long-range contacts. Actually, short- and medium-range NOEs are reporters of secondary structure, whereas long-range NOEs indicate the occurrence of a rigid tertiary structure. Moreover, unfolding of the heme pocket has been followed by electronic absorbance spectroscopy (Fig. 1BGo). Changes of tertiary and secondary structure and heme release are cooperative reversible processes. Data have been analyzed according to Equation 1Go; values of [urea]0.5, m, and {Delta}GU° (see Equation 1Go) are reported in Table 1Go.


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  		Table 1. Thermodynamic parameters for the urea-induced unfolding equilibrium of C. caretta Mba
 
By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained as it occurs at the helix interfaces. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered. It is worth noticing that this region is reported to be involved in the stabilization of a molten globule during cooperative unfolding of apoMb at acidic pH. On the other hand, helices C and D, as well as helix F, loose structural constraints at 3.0 M urea. The GH hairpin and helix E are still described by a sufficient number of structural contacts, and the diagram does not show significant changes in this region, as shown in Figure 1DGo. Also the reversible unfolding of ferric Aplysia limacina Mb involves a limited opening of the hemoprotein, owing to the displacement (hinge bending) of helix A (Janes et al. 1987).

At higher urea concentration (5 M), the secondary structure is no longer defined, because all NOE values are lost. Of course, this would not imply that the heme pocket is totally unfolded; rather, the loss of NOE constraints in this region does not allow us to draw a structural model at this urea concentration. At urea concentration >6 M, the protein appears to be fully unfolded, including the region stabilized by contacts with the prosthetic group.

Denaturation curves obtained by following long-range and short- to medium-range NOE peaks display different [urea]0.5 values from those obtained by monitoring electronic absorption changes (see Table 1Go). Present results indicate that the cyanide derivative of ferric C. caretta Mb undergoes reversible urea-induced partial denaturation, with a free energy change of 10.5 kJ/mole. Data shown in Figure 1BGo may be described accounting for the minimum three-state mechanism N{longleftrightarrow}X{longleftrightarrow}D, where X represents a disordered globin structure (occurring at ~4 M urea) that still binds the heme. For the partly denatured structure, residual NOEs are still sufficient to draw a diagram for the sequential unfolding of the globin domain, in agreement with a hierarchic mechanism (Freund et al. 1996; Baldwin and Rose 1999; Ragona et al. 1999). The transition to the fully denatured, heme-free state (D) is again reversible and fully cooperative, and occurs with a variation of the Gibbs free energy of 38.7 kJ/mole.


   Materials and methods
TOP
 Abstract
 Introduction
 Materials and methods
References
 
C. caretta myoglobin was prepared according to literature (Nardini et al. 1995). All chemicals were from Sigma Chemical Co. Fifteen milligrams of C. caretta Mb were dissolved in 0.5 mL of 50 mM phosphate buffer (pH 5.4), and 50 µL deuterium oxide were added to provide the field-frequency lock. After transfer to the NMR tube, 3.0 µg potassium cyanide were added to the sample.

DQF-COSY, TOCSY, and NOESY experiments were acquired on a Bruker AVANCE 600 NMR spectrometer (Bruker Analytik) equipped with a pulsed field gradients device. All experiments were acquired with two different spectral widths: 30,000 Hz (to assign frequencies belonging to protons near the paramagnetic center of the heme cavity) and 8000 Hz (to improve the resolution in the diamagnetic region) for both f1 and f2 dimensions. In the former case, the water signal was suppressed by low-power presaturation during the relaxation delay (1 sec); in the second case, by a 3-9-19 selective excitation with gradients (Sklenar et al. 1993). In all experiments, 352 scans were acquired per t1 increment; the mixing time was 40 msec for NOESY and 100 msec for TOCSY. Data were acquired as matrices of 1024(t2) x 512(t1) complex points and apodized with a square cosine window function. All NOESY and TOCSY experiments were performed by using the States-TPPI phase cycling scheme, whereas DQF-COSY experiments were performed by using the TPPI phase cycling scheme (Cavanagh et al. 1996). The f1 dimension was extended to 500 points by linear prediction before two-dimensional Fourier transformation.

Structural models have been obtained by restrained torsion-angle dynamics using DYANA (Güntert et al. 1997). Paramagnetic shifts arising from pseudocontact interaction between the low-spin Fe(III) center and the side-chain protons have been included in the model by means of the PSEUDYANA subroutine (Banci et al. 1997). Atomic coordinates of the models were visualized with the Swiss PDB Viewer (Guex and Peitsch 1997).

Electronic absorption spectra of C. caretta Mb were obtained in 1-cm pathlength cuvettes on a Beckman DU-640 spectrophotometer.

Reversible urea-induced denaturation data (see Fig. 1BGo) were analyzed according to Equation 1Go (Pace 1986).

((1))


   Acknowledgments
 
We acknowledge Professors M. Brunori, A. Desideri, and M. Rizzi for stimulating discussion, and the Bioindustry Park Canavese, Colleretto Giacosa, Italy, for providing access to the 600-MHz NMR facility.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
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 Introduction
 Materials and methods
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