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¹⁵N-¹H Residual Dipolar Coupling Analysis of Native and Alkaline-K79A Saccharomyces cerevisiae Cytochrome c

Michael Assfalg ^*, Ivano Bertini ^*, Paola Turano ^*, A. Grant Mauk , Jay R. Winkler  and Harry B. Gray 

^* Magnetic Resonance Center (CERM), University of Florence, 50019 Sesto Fiorentino, Florence, Italy; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; and Beckman Institute, California Institute of Technology, Pasadena, California 91125 USA

Correspondence: Address reprint requests to Prof. Ivano Bertini, CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy. Tel.: 39-055-457-4272; Fax: 39-055-457-4271; E-mail: bertini{at}cerm.unifi.it.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide ¹⁵N-¹H ¹J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Molecules with molecular magnetic anisotropy (^mol) tend to adopt preferred orientations in an external magnetic field (Lohman and Maclean, 1978; Domaille, 1980; Bothner-By et al., 1981, 1984, 1985). Partial alignments give rise to effects that are detectable through nuclear magnetic resonance (NMR) spectroscopy (Van Zijl et al., 1984; Bothner-By, 1996). Among these, the noncompletely averaged dipolar couplings or RDCs (Tolman et al., 1995; Tjandra and Bax, 1997) represent a source of structural information (Tjandra et al., 1997; Clore et al., 1998; Meiler et al., 2000; Sass et al., 2000). More recently, RDCs in different orienting media have provided information on dynamical properties (Meiler et al., 2001; Bax and Tjandra, 1997; Tolman et al., 1997).

Heme proteins possess molecular magnetic anisotropy (Tolman et al., 1997; Banci et al., 1998; Arnesano et al., 2000). In the iron(II) diamagnetic form, magnetic anisotropy arises from the heme, aromatic moieties, and elements of secondary structure (Banci et al., 1998; Arnesano et al., 2000). In an iron(III) S = heme, the molecular magnetic anisotropy arises from the diamagnetic contributions (^dia) and the dominant paramagnetic contribution of the iron(III) ion (^para) (Banci et al., 1998; Arnesano et al., 2000). The latter contribution can be evaluated independently through analysis of the ¹H pseudocontact shifts (PCs) (Williams et al., 1985; Emerson and La Mar, 1990; Banci et al., 1995, 1996; 1997a,b; Arnesano et al., 1998, 1999; Bentrop et al., 1997). In diamagnetic heme proteins, the magnetic anisotropy is small and RDCs are barely large enough to be used as structural constraints. In paramagnetic low-spin heme proteins, the RDCs are in the range -1.5 to 1.5 Hz (Tolman et al., 1997; Banci et al., 1998; Arnesano et al., 2000; Bertini et al., 2001a,b).

Mitochondrial cytochrome c is a heme protein containing diamagnetic iron(II) in its reduced form, and a paramagnetic S = iron(III) in its oxidized form. In both redox states the iron ion is axially coordinated by His and Met side chains (Berghuis and Brayer, 1992; Dickerson et al., 1971; Moore and Pettigrew, 1990; Baistrocchi et al., 1996; Banci et al., 1997a,b; Scott and Mauk, 1996). The Fe-Met bond is relatively labile in oxidized cytochrome c, and at high pH the native methionine ligand is replaced either by Lys73 or Lys79, giving the so-called alkaline form of the protein (Wilson and Greenwood, 1996; Assfalg et al., 2003). We report here RDCs for both the oxidized and reduced states of cytochrome c (cyt c) at pH 7.0. These RDCs have been included in solution structure calculations together with other available constraints (Baistrocchi et al., 1996; Banci et al., 1997a,b). For the non-native form of the oxidized protein at pH 11.1, both RDCs and PCs have been estimated. From this analysis, an extensive mobility is revealed at high pH.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Sample preparation
NMR measurements on reduced cytochrome c were made on the yeast protein expressed in Escherichia coli (Barker et al., 2001); the protein contains, at the N-terminus, a tag of three residues—A, D, and L. K79A cyt c from Saccharomyces cerevisiae was expressed and purified as previously reported (Assfalg et al., 2003). This sample was used to measure the RDCs for ferricytochrome c at pH 7.0 and for the alkaline form. The use of different mutants for measurements on the reduced and oxidized proteins at pH 7.0 is justified by the essential identity of the NMR spectra of these cyt c variants, as demonstrated previously (Assfalg et al., 2003; Barker et al., 2001). The NMR samples were prepared by dissolving the protein in 50 mM phosphate buffer at pH 7.0 to give solutions 2 mM in protein. For the alkaline form, the pH was increased by addition of NaOH to give pH 11.1.

NMR spectroscopy
All NMR experiments were acquired at 298 K on Avance500 and Avance800 spectrometers (Bruker, Karlsruhe, Germany).

Twenty ¹J_NH-modulated 2D ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra were recorded at 500 and 800 MHz for Fe(II)-, Fe(III)-, and alkaline Fe(III)-cyt c. Dephasing delays, 2, were 43.6, 45.0, 46.4, 47.8, 49.2, 50.6, 52.0, 52.6, 53.4, 54.8, 55.4, 56.2, 56.8, 57.6, 59.0, 60.4, 61.8, 63.2, and 64.6 ms. These delays included the durations of the pulsed field gradients, G4, but not the duration of the ¹⁵N 180° pulse. The intensities of cross peaks are given by Eq. 1 (Tjandra et al., 1996):

(1)

where I₀ is the intensity of the cross peak when is null, A is a term which accounts for the unmodulated fraction of magnetization due to pulse imperfection, and 1/T₂^* is the effective decay rate of the transverse ¹⁵N magnetization. As previously suggested (Tjandra et al., 1996), 2 values should be chosen as two sets of symmetric values (2n+1)/2J, where n is an integer, to take into account effects of 180° ¹⁵N pulse imperfections and to optimize the accuracy of the measurement of J. The best value of n, which depends on the value of T₂^*, was chosen after measuring T₂^* of some cross peaks from the ¹J_NH-modulated HSQC spectra acquired with delays of 5.4, 10.8, and 21.6 ms. These delays correspond to 1/2J, 1/J, and 2/J, and yield almost completely refocused intensity for all NH moieties. Therefore, for these delays the HSQC cross-peak intensities are only dependent on the transverse relaxation of ¹⁵N spins. An average T₂^* value of 40 ms was found; n was thus set as 4 and 5. Each experiment was performed with 16 scans.

{¹H}-¹⁵N nuclear Overhauser effect (NOE) experiments were performed both at 400 MHz and 700 MHz. The pulse sequence is the one previously described (Grzesiek and Bax, 1993; Palmer III et al., 1992; Barbato et al., 1992; Kay et al., 1992; Grzesiek and Bax, 1993; Peng and Wagner, 1992), which includes sensitivity enhancement and water suppression with the flip-back method. {¹H}-¹⁵N NOE saturated and unsaturated spectra were acquired in an interleaved fashion, with 40 scans for the 700 MHz measurements and 128 scans for the 400 MHz experiments. Phase sensitivity was achieved using Echo/Antiecho-TPPI gradient selection. A recycle delay of 3.1 s was used. 2048 complex data points with 512 complex increments were collected.

Data processing and integration of cross peaks was performed on a Silicon Graphics workstation using standard Bruker (Karlsruhe, Germany) software. Rectangular boxes at the noise level were used to define the integration region, except for overlapping cross peaks, for which smaller boxes were chosen to minimize the contribution from the unwanted peak. The obtained values were fitted to Eq. 1 with a Levenburg-Marquardt algorithm to obtain ¹J_HN values. Experimental errors were estimated by Monte Carlo methods.

Solution structure calculations with RDC constraints
The solution structure calculations were performed with the PARAMAGNETIC-DYANA program (Barbieri et al., 2002; information on program available at www.postgenomicnmr.net). Initial values for the molecular magnetic tensor parameters were obtained with the program FantaOrient (Banci et al., 1998; information on program available at www.postgenomicnmr.net). For each protein, 500 random structures were annealed in 10,000 steps by introducing the available NOE and PCs (Fe(III)-cyt c) constraints, together with the RDC constraints. The relative weights of all constraints were set equal to 1 and the tolerance values T_i for RDC were taken equal to the estimated measurement errors. The 20 best structures (i.e., the ones with the lowest total target function value) of the new family of conformers were used to re-estimate the tensor parameters for a new refinement step. The calculations were repeated until convergence of the tensor parameters was reached.

Determination of the magnetic susceptibility anisotropy
The molecular magnetic susceptibility tensor can be obtained from the difference in RDC values, RDC, measured at two different fields. The RDC values are given by the difference between the ¹J_HN values measured at the two different fields, corrected for the contribution due to the diamagnetic dynamic frequency shift (_DFS), which constitutes a non-negligible magnetic field-dependent contribution to apparent ¹H-¹⁵N ¹J (Tjandra, Grzesiek, and Bax, 1996).

RDC values are given by Eq. 2 (Van Zijl et al., 1984):

(2)

where _ax^mol and _rh^mol are, respectively, the axial and rhombic components of the molecular tensor, ^mol, , and are polar coordinates describing the orientation of the N-H bond vector in the (axis) frame of the ^mol tensor, and S is the generalized order parameter. This form of the equation allows RDC to be expressed as a function of the molecular ^mol tensor, taking into account the relationship between it and the orientation tensor (Bertini et al., 2002). The tensor parameters were obtained with the program FantaOrient (Banci et al., 1998), which optimizes the fit of experimental RDC values over the given structure. The adjustable parameters are _ax^mol and _rh^mol, as well as the three independent direction cosines needed to define the orientation of the ^mol tensor within the laboratory frame.

An important contribution to the molecular magnetic susceptibility in paramagnetic proteins is the paramagnetic susceptibility, which is traditionally evaluated from pseudocontact shifts (Emerson and La Mar, 1990; Banci et al., 1992, 1996), according to Eq. 3, which holds within the metal-centered point-dipole/point-dipole approximation:

(3)

where _ax^para and _rh^para are axial and rhombic anisotropies of the magnetic susceptibility tensor, and are cylindrical coordinates of the position vector of a proton, and r is the distance between the paramagnetic center and the proton in question.

Fits of the pseudocontact shifts over the structure employed the program Fantasian (available from http://www.postgenomicnmr.net), following a procedure analogous to that used with RDC data.

Determination of residual dipolar splittings is an alternative to the use of dipolar chemical shifts for the study of the metal-center magnetism in paramagnetic proteins (Déméné et al., 2000; Tolman et al., 1995; Banci et al., 1998). The magnetic susceptibility tensor can be derived from the difference between the corresponding ¹J_HN values for the paramagnetic and diamagnetic species, measured at the same field, according to Eq. 4:

(4)

with symbols defined as in Eqs. 2 and 3.

The following quantities were obtained (Eq. 5):

(5)

The magnetic anisotropy tensor was evaluated by obtaining best fits of experimental RDC^para values over a refined family of structures with the FantaOrient program.

Monte Carlo methods were used to make error estimates for all tensor parameters: 200 different calculations were performed on data sets, where 30% of experimental values were randomly eliminated from input data.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The case of oxidized-K79A and reduced cytochrome c and solution structure refinements
The backbone ¹⁵N-¹H ¹J values were measured at 500 and 800 MHz for Fe(III)-cyt c and Fe(II)-cyt c at pH 7.0. RDC^800-500 values for 54 and 73 ¹⁵N-¹H pairs were obtained for the two species (Fig. 1, A–B). The values range between 0.78 and -0.62 for Fe(II)-cyt c, and 1.46 and -1.41 for Fe(III)-cyt c.

View larger version (21K): [in this window] [in a new window]   FIGURE 1  RDC800–500 values measured for (A) Fe(II)-cyt c, (B) Fe(III)-cyt c, and (C) alkaline K79A Fe(III)-cyt c. The residue numbering starts at -5, as is common in the cytochrome-c literature; tuna cyt c is the reference, allowing conserved residues to have the same numbers in all mitochondrial cytochromes c.

View larger version (13K): [in this window] [in a new window]   FIGURE 2  (A) Observed vs. calculated RDCpara values for the refined best structure of Fe(III)-cyt c. (B) Observed vs. calculated RDC800–500 values for the refined best structure of Fe(II)-cyt c.

View this table: [in this window] [in a new window]   TABLE 1  Parameters characterizing the para, mol, and dia tensors as obtained from the PCs and the RDC of Fe(III)-cyt c and the RDC of Fe(II)-cyt c

The case of alkaline-K79A-Fe(III)-cyt c and characterization of mobility
The RDC^800–500 values measured for Fe(III)-cyt c at pH 11.1 span a range smaller than that of oxidized protein at pH 7.0; i.e., their values are in the range +0.88 to –0.92 (Fig. 1 C). This is particularly true for residues -5 to 42 and 79 to 103. This is quite surprising if one applies the same analysis reported above for oxidized Fe(III)-cyt c at pH 7. Indeed, the ^para values estimated from PCs indicate that the alkaline form of Fe(III)-cyt c is characterized by a _ax^para even larger than that of Fe(III)-cyt c at pH 7.0 (Table 2). The PCs-derived magnetic tensor parameters of ^para determined for the alkaline form are in excellent agreement with those already reported by us for cyt c-CN^- and cyt c-NH₃, i.e., derivatives where the sixth ligand of the low-spin iron(III) has cylindrical symmetry and does not contribute to the rhombic component of ^para. On the other hand, the observed difference in RDC^800–500 values between the native and alkaline forms cannot be accounted for by any difference in ^dia that could be induced by differences in backbone conformation. Indeed, in globular proteins the protein backbone contribution is essentially negligible (see above). Moreover, we have determined a solution structure for the alkaline Fe(III) protein based on NOE and PCs constraints (Assfalg et al., 2003), and the observed differences do not justify significantly different ^dia values. We therefore propose that the reduction in RDCs arises from increased mobility of the pH 11.1 conformer relative to the native structure, an explanation that is consistent with the reduced number of NOE cross peaks detectable in the NOE spectroscopy (NOESY) spectra of the alkaline form with respect to the corresponding spectra of native Fe(II) and Fe(III) cyt c (500 fewer NOEs were used in alkaline-cyt c structure calculations). Indeed, the presence of internal motions faster than the overall molecular tumbling reduces the NOE intensity. To have direct information on protein mobility, heteronuclear relaxation measurements and {¹H}-¹⁵N NOE experiments were performed. The unavoidable local overheating of the sample during R₂ measurements causes protein precipitation under the extreme pH conditions, making this experiment meaningless. Information on the effective correlation time was therefore obtained through analysis of the field-dependence of the {¹H}-¹⁵N NOE intensity. Reliable values of {¹H}-¹⁵N NOE, measured at 700 MHz, were obtained for 67 backbone HN resonances and were found to range from 0.56 to 0.97. The calculated average NOE value was 0.80 ± 0.09. From the measurements performed at 400 MHz, the NOEs of 62 resonances were found to range from 0.30 to 0.92, with an average value of 0.66 ± 0.17. An analysis of NOE intensities at two fields provides an estimate of the effective correlation time for nuclear reorientations. From the above values, an average effective correlation time of 4.0 ns is obtained, which drops to 3.0 ns for loop residues. In this range of correlation times, the measurements at 400 MHz become particularly useful, because small changes in effective _c values produce significant variations in NOEs, as compared with the small variations observed at 700 MHz. The effective correlation time is clearly shorter than the 5.2 ns measured for the native protein under neutral conditions (Fetrow and Baxter, 1999), indicating the presence of substantial mobility at high pH.

View this table: [in this window] [in a new window]   TABLE 2  Parameters characterizing the para and mol tensors as obtained from the PCs and the RDCs of alkaline K79A Fe(III)-cyt c

Paramagnetic systems are ideal because 1), they do not need external orienting devices that may be problematic under extreme pH conditions, and 2), they provide a reliable estimate of ^para, showing that partial orientation indeed occurs. The fact that pseudocontact shifts are not quenched by mobility that quenches RDCs indicates that the motions maintain, on average, the same metal-to-proton distances. Indeed, the orientational average of an NH vector may reduce RDCs, whereas the average polar coordinates of an amide proton, which determine the pseudocontact shift, may vary only a little. The situation is summarized in Fig. 3; a change in the orientation of an NH bond determines a sensible variation in the angle between the NH vector and the magnetic axis, whereas the angle between the same axis and the metal-H vector is only slightly affected, as in the case of the alkaline form of K79A cytochrome c.

View larger version (13K): [in this window] [in a new window]   FIGURE 3  Mobility of amide protons affects the RDCpara and PCs values to a different extent. A change in the position of the proton determines a larger change in the angle RDC than in the angle PCs because the metal-proton distance is larger than the nitrogen-proton distance.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

For both Fe(III)-cyt c and Fe(II)-cyt c at neutral pH, RDC values were used together with available NOEs (and PCs shift for the oxidized protein) to recalculate solution structures. Although the structures are essentially the same as before, they are more reliable, as they are consistent with two or three (NOEs, PCs, and RDCs in the case of Fe(III)-cyt c) sets of constraints. From the structures, the molecular magnetic anisotropies and directions are consistent with the values of the metal ion magnetic susceptibility anisotropy obtained from pseudocontact shifts.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
We thank Federico Rosell for providing samples of K79A cytochrome c.

We acknowledge support from the Ministero dell'Istruzione, dell'Università e della Ricerca, Cofinanziamento 2001 MIUR COFIN2001) and European Community Network (grant FMRX-CT98-0218 to I.B.); Italian Consiglio Nazionale delle Richerche (Progetto Finalizzato Biotecnologie grant 01.00359.PF49 to P.T.); operating grant MT-14021 from the Canadian Institutes of Health Research and a Canada Research Chair (to A.G.M.); United States National Institutes of Health grant DK19038 (to H.B.G.); and Department of Energy grant DE-FG03-02ER15359 (to J.R.W.).

Submitted on September 27, 2002; accepted for publication January 21, 2003.

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