Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform

Na Sun, An Wang, Aaron B. Cowley, Adriana Altuve, Mario Rivera and David R. Benson1

Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045-7582, USA

1 To whom correspondence should be addressed. E-mail: drb{at}ku.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the McS71L b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.

Keywords: apoprotein/cytochrome b5/heme replacement/mutagenesis/stability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The two genes coding for membrane-associated isoforms of cytochrome b5 in mammals (Lederer et al., 1983Go) probably arose via duplication of a primordial gene (Guzov et al., 1996Go). Subsequent functional divergence resulted in targeting of one isoform to the membrane of the endoplasmic reticulum (microsomal or Mc b5) and of the other to the outer mitochondrial membrane (OM b5) (Mitoma and Ito, 1992Go; Kuroda et al., 1998Go). Mc and OM b5 are anchored to membranes of their respective organelles by a C-terminal hydrophobic domain, with their soluble heme-binding domains extending into solvent (Ozols, 1989Go; Vergeres and Waskell, 1995Go). Research in our laboratories has shown that Mc and OM b5 heme-binding domains exhibit markedly divergent biophysical properties (Altuve et al., 2004Go), consistent with involvement in different physiological processes (Vergeres and Waskell, 1995Go; Soucy and Luu-The, 2002Go; Ogishima et al., 2003Go).

Storch and Daggett reported molecular dynamics (MD) simulations showing that the heme-binding domain of bovine Mc b5 exhibits substantial polypeptide conformational mobility (Storch and Daggett, 1995Go). They suggested that this might reflect a need for adaptability in recognition of diverse redox partners, but that it could also represent initial stages of polypeptide unfolding. Evidence supporting a relationship between polypeptide dynamic mobility and b5 stability has been provided by studies in our laboratories comparing recombinant proteins representing the heme-binding domains of bovine Mc (bMc) b5 and rat OM b5 (rOM b5) (see Figure 1). Hydrogen–deuterium exchange (HDX) experiments monitored by NMR spectroscopy have shown that rOM b5 exhibits dampened polypeptide dynamic mobility in comparison with bMc b5 (Simeonov et al., 2005Go), confirming and extending observations in earlier MD simulations (Altuve et al., 2001Go; Lee and Kuczera, 2003Go). In addition, OM b5s are kinetically inert with respect to dissociation of hemin (ferric heme) at 37°C and pH 7, conditions under which loss of hemin from Mc b5s to apomyoglobin (apoMb) is complete within a couple of days (Silchenko et al., 2000Go; Altuve et al., 2004Go). It is reasonable to assume that OM b5s also exhibit enhanced thermodynamic stability relative to Mc b5s under these physiologically relevant conditions. Providing proof of this possibility has been hampered by factors such as incomplete reversibility of unfolding reactions, however (Cowley et al., 2002Go).



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Fig. 1. Amino acid sequences of the recombinant bMc and rOM b5 heme-binding domains used in the present work. Residues are numbered according to the scheme originally proposed for the lipase fragment of bMc b5 (Mathews et al., 1979Go). Heme ligands His39 and His63 are shown in bold. The letters under the rOM b5 sequence indicate residues with side chains participating in conserved OM b5 hydrophobic packing motifs a and b.

 
Mc and OM b5 heme-binding domains exhibit essentially identical three-dimensional folds comprising two hydrophobic cores (Rodriguez-Maranon et al., 1996Go), as highlighted in Figure 2A by the X-ray crystal structure of bMc b5 (Durley and Mathews, 1996). Heme resides in core 1, stabilized by (1) ligation between iron and the side chains of His-39 and His-63; (2) hydrogen bonds between Ser-64 and a heme propionate group (Lee et al., 1991Go; Hunter et al., 1997Go); and (3) non-specific interactions between the protoporphyrin IX (PPIX) moiety and predominantly apolar amino acid side chains from the surrounding four-helix bundle and the core 1 side of the ß-sheet. Because substituents on PPIX are asymmetrically distributed, all known b5s exist in two diastereomeric forms differing by a 180° rotation about the PPIX {alpha}-{gamma}-meso axis. The thermodynamically dictated isomer ratio is governed in significant measure by the steric environment provided by amino acid side chains lining the base of the heme binding pocket (Lee et al., 1990Go, 1991Go; Mortuza and Whitford, 1997; Altuve et al., 2001Go; Cowley et al., 2002Go). The orientations are designated A and B, with A dominating at equilibrium in all known Mc b5s (A:B ~ 9:1 in bMc b5) (McLachlan et al., 1986Go). OM b5s exhibit a slight preference for hemin orientation B (A:B = 1:1.2 in rOM b5 (Silchenko et al., 2000Go)). Amino acid side chains contributing to these differences are highlighted in Figures 2B and 2C.



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Fig. 2. (A) Representation of the X-ray crystal structure of bMc b5 (PDB 1CYO) (Durley and Mathews, 1996). (B) View of the bMc b5 structure highlighting residues involved in van der Waals contact with hemin substituents on pyrrole rings I and II (orientation A). (C) Corresponding view of the rOM b5 crystal structure, in which hemin was modeled in orientation A (PDB 1B5M) (Rodriguez-Maranon et al., 1996Go).

 
Removal of heme from Mc and OM b5 causes essentially complete disruption of secondary and tertiary structure in core 1, but leaves core 2 and strands 1–4 of the ß-sheet with significant holo-like structure (Huntley and Strittmatter, 1972; Falzone et al., 1996Go; Falzone et al., 2001Go; Cowley et al., 2004Go). The resulting apoproteins (bMc and rOM b5 apo-b5) exhibit similar thermodynamic stability under physiologically relevant conditions (Silchenko et al., 2000Go; Cowley et al., 2004Go), in stark contrast to observations noted above for the corresponding holoproteins. Recent studies indicated the empty rOM apo-b5 heme binding pocket to be more compact and less dynamically mobile than that of bMc apo-b5, however (Cowley et al., 2004Go), which led to the following conclusions: (1) the lower hemin release barriers of Mc b5s in comparison to OM b5s results not only from more frequent conformational changes that can trigger prosthetic group dissociation, but also from the fact that a given triggering event will tend to be accompanied by more extensive melting of local structure; and (2) the apparently greater thermodynamic stability of OM holo-b5s in comparison to Mc holo-b5s is at least partially due to a smaller entropic penalty for polypeptide folding associated with heme binding.

We have identified two conserved hydrophobic packing motifs (Motifs a and b) near the core 1/core 2 interface in OM b5s, which are more extensive than the corresponding conserved Mc b5 interactions (Altuve et al., 2004Go). Residues contributing to these different conserved interactions in rOM and bMc b5 are highlighted in Figure 1 and some also appear in Figures 2B and 2C. Studies in which amino acids contributing to rOM b5 Motifs a (Altuve et al., 2001Go) and b (Cowley et al., 2002Go) are systematically replaced by the corresponding Mc b5 residues have shown that they contribute to the higher OM b5 hemin release barriers. Replacing Motif a in its entirety via the A18S/I32L/L47R triple mutant caused a modest decrease in holoprotein stability, without significantly affecting apoprotein stability. More recently we have shown that the Motif a swap exerts little if any effect on core 2 structure or core 1 compactness in rOM apo-b5 (Cowley et al., 2005). On the other hand, replacing Leu-71 in rOM b5 Motif b with Ser led to large decreases in both holo- and apoprotein stability (Cowley et al., 2002Go). It also resulted in a large increase in polypeptide expansion that accompanies heme release, well beyond that observed for bMc b5, in large part because it caused apoprotein conformational disorder to extend beyond core 1 and into core 2 (Cowley et al., 2005).

The results of the studies described above suggested to us that Leu-71 plays a dominant role in maintaining the empty heme-binding pocket of OM apo-b5 in a compact state relative to that of Mc apo-b5. As a means of probing this possibility and testing a hypothesis that it contributes to the higher hemin release barriers exhibited by OM holo-b5s relative to Mc holo-b5s, we generated the S71L mutant of bMc b5. As described in this report, the S71L mutation increased the compactness of the empty bMc apo-b5 heme-binding pocket. It also resulted in a diminished hemin release rate constant, but only for the isomer with hemin in orientation B. In contrast, the S71L mutation accelerated release of hemin from orientation A. Steric strain between hemin and the side chains of Leu-71 and other amino acids at the base of its binding pocket as a source for this observation was suggested by broadening of signals for hemin substituents in 1H NMR spectra of the former. This hypothesis was confirmed in studies comparing wild-type bMc b5 (bMcWT b5) and its S71L mutant (bMcS71L b5) in which hemin had been replaced by the less sterically demanding analogue deuterohemin, as well as by introduction of a second rational mutation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Site-directed mutagenesis

The QuikChange site-directed mutagenesis kit (Stratagene) was used to construct the S71L and L32I/S71L mutants of bMc b5, starting from the pET 11a plasmid harboring (Cowley et al., 2002Go) the synthetic gene (Funk et al., 1990Go) for the wild-type protein. The primers used to introduce the S71L mutation were 5'-GTACCGACGCTCGTGAACTGCTGAAAACGTTCATCATCGGTG-3' (forward) and 5'-CATGGCTGCGAGCACTTGACGACTTTTGCAAGTAGTAGCCAC-3' (reverse). The primers used to introduce the L32I mutation were 5'-GTATACGACATAACTAAATTCCTGGAAGAGCACC-3' (forward) and 5'-TTTAGTTATGTCGTATACTTTGTAGTGCAGGATCAG-3' (reverse). The underlined codons indicate the site at which the mutations were introduced. The recombinant constructs were transformed into Escherichia coli XL1 blue competent cells for amplification. Once the mutations were confirmed by gene sequencing, the recombinant plasmids were transformed into E.coli BL21(DE3) cells for protein expression.

Proteins

Proteins were expressed and purified as described previously (Rivera et al., 1992Go), with the following changes introduced to enhance the yield of holoprotein. (1) Expressions were performed at 27°C rather than at 37°C, as the lower temperature has been shown to mitigate formation of inclusion bodies by bMc apo-b5 and to generally enhance yields of holoproteins in all cases (Cowley et al., 2004Go). (2) Hemin was added to the crude supernatant to convert residual apoprotein to the holo form (Falzone et al., 1996Go), followed by centrifugation to remove excess hemin. Concentrations of holoprotein in experimental samples were estimated by electronic absorption spectroscopy, using the absorbance at 412 nm (Soret band {lambda}max) and an assumed extinction coefficient ({varepsilon}412) of 130 000 M–1 cm–1 (Beck von Bodman et al., 1986Go). Apoproteins were prepared using the acid butanone method of Teale (Teale, 1959Go) and the stock solutions were maintained at 4°C and used within 2 days of preparation. Apoprotein concentrations were determined from the extinction coefficient at 280 nm ({varepsilon}280 = 11 460 M–1 cm–1 for bMcWT, bMcS71L and bMcL32I/S71L apo-b5; 12 950 M–1 cm–1 for rOM apo-b5), calculated based on the number of Trp and Tyr residues (Gill and von Hippel, 1989).

Analytical expression experiments

Analytical expression experiments (Cowley et al., 2004Go) with rOM, bMcWT and bMcS71L bMc b5 were always performed simultaneously using the same protocol as for preparative runs, but with 250 ml of LB medium. IPTG was added to each sample only when its OD600 had reached a value of 1.0 (~5 h). Identical solution volumes were maintained throughout the lysis and centrifugation steps. Actual yields of holoproteins were calculated by electronic absorption spectroscopic analysis of supernatants, using the absorbance at 412 nm. Relative amounts of apoproteins were evaluated by densitometric analysis of native PAGE gels (Bio-Rad Molecular Imager FX; Table I). Table I reports data obtained in four separate trials for the holoproteins and in two separate trials for the apoproteins. Cell pellets were analyzed for inclusion bodies using an established method (De Bernardez Clark, 1998Go).


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Table I. Data from analytical expression and DLS experiments

 
Dynamic light scattering (DLS)

DLS measurements (Schmitz, 1990Go) were performed on a BI-200SM research goniometer with a BI-9000AT digital correlator (Brookhaven Instruments). Incident light of {lambda} = 532 nm (0.3–1.0 W) was used, with scattered light detected with a photomultiplier tube at an angle of 90°. Sample temperature was controlled by means of a thermostated cell jacket and monitored with a thermocouple. Samples (100 mM) were passed through 100 nm filters (Whatman) immediately before each use. Two independent measurements were performed for each protein, each consisting of six 30 s runs which were averaged by the instrument software to obtain standard deviations. All data could be fitted multimodally and essentially 100% of the scattering mass was attributed to a single low molecular mass component. The diffusion coefficient (D) and the hydrodynamic radius (Rh) are related by the Stokes–Einstein equation:

(1)
where T is the temperature in kelvin, k is the Boltzmann constant (1.38 x 10–16 erg/K) and {eta} is the solution viscosity (set at 1.0 and 1.663 g/cm.s for samples examined in buffered aqueous solution and in 8 M urea, respectively, at 25°C) (Kawahara and Tanford, 1966).

Circular dichroism (CD) spectroscopy

CD spectra were recorded on a Jasco J-710 spectropolarimeter equipped with a Jasco PTC-4235 Peltier thermostated cell holder. A 0.2 cm flat cell was used in all cases. Far-UV (190–250 nm) and near-UV (255–340 nm) spectra were acquired at 1.0 nm intervals with a response time of 4 s and a scan rate of 50 nm/min and represent the average of five scans.

Chemical denaturation studies

All chemical denaturation experiments were performed at 25°C, with solutions buffered to pH 7.0 using 30 mM MOPS. Denaturant stock solution concentrations were verified from measurements of the solution refractive index. All samples were incubated at 25°C for 1 h before spectra were recorded. Holoprotein chemical denaturation was monitored by electronic absorption spectroscopy using a Varian Carey 100 Bio UV–visible spectrophotometer equipped with Peltier-thermostated cell holder (protein concentrations of 3–4 µM), following the change in absorbance at 412 nm (Soret band {lambda}max). Apoprotein chemical denaturation was monitored by fluorescence spectroscopy using a PTI QuantaMaster luminescence spectrometer (protein concentrations 0.5–1 mM), following changes in emission (340 nm) of Trp22 (excitation at 295 nm). Apoprotein denaturation curves were fitted (Kaleidagraph, v. 3.5) to the equation (Pace, 1986Go)

(2)
where {Delta}GN->U is the free energy of unfolding in the absence of denaturant, [D] is the concentration of denaturant and m is a parameter indicating the sensitivity of the free energy of unfolding to denaturant concentration. The concentration of denaturant at which each protein is 50% denatured (Cm value) was calculated using the equation

(3)

Mean values and average deviations of {Delta}GN->U, Cm and m calculated from three independent runs for each apoprotein are reported in Table II. Because holoprotein denaturation is not fully reversible and also appears to deviate from two-state behavior (Cowley et al., 2002Go), Table II reports only mean values and average deviations of Cm obtained from the inflection points in denaturation curves obtained in three independent runs.


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Table II. Chemical denaturation and heme transfer data

 
Hemin transfer studies

Horse skeletal apomyoglobin (apoMb) was employed as the receiving protein in hemin transfer experiments. Removal of heme from the holoprotein (Calbiochem) was accomplished using the method of Teale (Teale, 1959Go). Measurements were performed at 37°C in 150 mM potassium phosphate solution buffered to pH 7.0, using a 20-fold excess of apoMb. Solutions contained 450 mM sucrose to help stabilize the apoproteins (Hargrove et al., 1994Go). ApoMb concentrations were estimated using an extinction coefficient of 16.0 mM–1 cm–1 at 280 nm (Harrison and Blout, 1965Go). The absorbance at 406 nm (A406) was monitored as a function of time and rate constants were determined by fits to a single exponential equation.

1H NMR spectroscopy

All samples (~1.0 mM protein) were prepared in perdeuterated sodium phosphate buffer (µ = 0.1 M, pH 7.0) to a final volume of ~500 ml; the pH values were not corrected for the isotope effect. 1H NMR spectra were acquired on a Varian Unity spectrometer operating at 598.658 MHz (1H frequency). Spectra were acquired with water presaturation over a 35 kHz spectral width, using a 0.4 s acquisition time and a 1.0 s relaxation delay.

Prosthetic group reconstitution

Experiments in which apoproteins were reconstituted with hemin (FeIII protoporphyrin IX chloride; FePPIX) or deuterohemin (FeIII deuteroporphyrin IX chloride; FeDPIX) were performed at 4°C. The stoichiometric amount of FePPIX or FeDPIX that was needed was calculated with the aid of a reconstitution experiment monitored by electronic absorption spectroscopy. Thus, a solution of freshly prepared apoprotein was exchanged into perdeuterated phosphate buffer (pH 7.0, m = 0.1) and concentrated to a final volume of ~500 ml (~1 mM). A small aliquot (5 ml) of this protein solution was diluted to 500 ml with phosphate buffer in a cuvette and titrated with a solution of FePPIX or FeDPIX obtained by diluting a 100 µl aliquot of a stock solution (10 mg/ml in DMSO-d6) to 300 µl with DMSO. The progress of the reconstitution was monitored by plotting the absorbance at the Soret band {lambda}max (412 nm for FePPIX; 402 nm for FeDPIX) as a function of the volume of stock solution added. The absorbance increases linearly with the addition of each aliquot of hemin until a stoichiometric amount has been added. Further addition of DPIX or PPIX results in a more gradual increase in absorbance at the Soret band {lambda}max; the inflection point in the resulting curve was used to calculate the volume of hemin equivalent to a 1:1 stoichiometric ratio. In reconstitutions with DPIX, 1.0 equivalent was added and the sample was subsequently passed through a G25 column to remove excess DPIX. Reconstitutions with PPIX were for use in hemin isomer equilibration studies monitored by NMR spectroscopy. In order to avoid equilibration prior to beginning the experiments and to limit problems due to excess hemin, 0.9 equivalent of hemin was added to the 500 ml solution of apo-b5. Rate constants for hemin–isomer interconversion were obtained by monitoring the time-dependent changes in the areas under the hemin resonances corresponding to isomers A and B as described previously (LaMar et al., 1984Go; Walker et al., 1988Go). The equilibrium constant was obtained from an NMR spectrum recorded after the isomer ratio had reached a constant value.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Effect of heme binding pocket compactness on polypeptide expression

The synthetic genes coding for the rOM (Rivera et al., 1992Go) and bMcWT b5 (Funk et al., 1990Go) polypeptides used in our studies (see amino acid sequences in Figure 1) are both under control of the strong T7 promoter in pET11a (Cowley et al., 2002Go). We have observed that bMcWT and rOM b5 polypeptide production in E.coli BL21(DE3) cells outpaces heme incorporation (Cowley et al., 2004Go), as previously reported for rat Mc b5 using a similar expression system (Falzone et al., 1996Go). The holo and apo forms of the two proteins in supernatants obtained after expression and cell lysis can be observed by native PAGE (Figure 3). Despite the use of analogous expression systems, rOM holo-b5 and apo-b5 are reproducibly obtained in at least 3-fold greater yield than the corresponding forms of bMcWT b5 when expressions are performed at 27°C (Table I), a temperature at which neither protein forms inclusion bodies (Cowley et al., 2004Go).



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Fig. 3. Native PAGE data for bMcWT, bMcS71L and rOMWT b5. Lanes 1–3, purified apoproteins; lanes 4–6, supernatants from analytical expressions.

 
Replacing Ser71 with Leu in bMcWT b5 substantially increases holoprotein production at 27°C and results in an increased level of apoprotein present in crude supernatants (Figure 3, Table I). In fact, the total yield of bMcS71L b5 polypeptide (holo plus apo) is similar to that observed for rOM b5 using identical expression conditions (Figure 3; Table I). In addition, the native PAGE data in Figure 3 show that bMcS71L apo-b5 migrates more rapidly than bMcWT apo-b5, consistent with our hypothesis that the S71L mutation would increase the compactness of the empty bMcWT apo-b5 heme-binding pocket. The S71L mutant apoprotein even migrates somewhat faster than rOM apo-b5, probably reflecting its smaller net negative charge (theoretical pI = 4.94 vs 4.62 for rOM apo-b5) rather than greater compactness of its binding pocket. Consistent with this interpretation, DLS data (Figure 4; Table I) show that removal of heme from bMcS71L apo-b5 results in a much smaller increase in hydrodynamic radius (4.3%) than observed for bMcWT b5 (9.8%), but still larger than occurs for rOM b5 (2.8%) (Cowley et al., 2004Go). It is noteworthy that bMcS71L apo-b5 also exhibits a polydispersity value (6.3%) midway between those of bMcWT (7.3%) and rOM apo-b5 (5.3%), suggesting an inverse correlation between core 1 compactness and the number of conformational states that it can explore. It is interesting to speculate that the large and highly reproducible increase in bMc b5 polypeptide production in our E.coli expression system resulting from the S71L mutation is related to the increase in heme-binding compactness indicated by the native-PAGE and DLS data. Perhaps increasing the compactness of structurally disordered core 1 protects it against proteolytic degradation.



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Fig. 4. Size distribution plots comparing DLS data for the holo (solid lines) and apo (dashed lines) forms of bMcWT b5 (left), bMcS71L b5 (center) and rOMWT b5 (right).

 
Enhanced compactness of bMcS71L apo-b5 relative to bMcWT apo-b5 is not associated with an increase in secondary structure, as we observe no change in the far-UV CD spectra (data not shown). The near-UV CD spectra of wild-type and bMcS71L apo-b5 are also virtually identical, indicating no change in tertiary structure. These observations are consistent with the fact that the mutation involves a residue in core 1, which is largely devoid of regular secondary structure to begin with and therefore also lacks organized packing in the vicinity of aromatic side chains located in that region.

The S71L mutation inverts the relative stabilities of hemin orientations A and B

The potential impact of the S71L mutation on bMcWT apo-b5 thermodynamic stability at 25°C and pH 7 was probed in guanidinium chloride (GdmCl)-mediated denaturation studies, monitored by fluorescence spectroscopy (Figure S1A, Supplementary data available at PEDS Online). Unfolding free energies for bMcWT and bMcS71L apo-b5 in the absence of GdmCl ({Delta}GN->U values), extrapolated from fits of the denaturation data to a two-state equation (Equation 2), are compiled in Table II. Table II also reports the concentration of GdmCl at which each apoprotein is 50% unfolded (Cm value), as well as {Delta}GN->U and Cm values previously reported for GdmCl-mediated denaturation of rOM apo-b5 (Cowley et al., 2002Go). Analogous studies with the holoproteins utilizing the more powerful denaturant GdmSCN were monitored by UV–visible spectrophotometry (Figure S1B; Table II). Because holo-b5 unfolding reactions are not fully reversible and tend to deviate from two-state behavior (Cowley et al., 2002Go), Table II only reports approximate Cm values for the holoproteins.

The S71L mutation led to a small increase in the Cm value of bMcWT holo-b5 (from 1.52 to 1.59 M GdmSCN), suggesting perhaps some increase in stability in the absence of denaturant. The mutation led to a considerably larger increase in the Cm value of the corresponding apoprotein (from 1.78 to 2.30 M GdmCl) and increased the unfolding free energy of bMcWT apo-b5 in the absence of denaturant by ~0.8 kcal/mol. It is important to emphasize that the difference in unfolding free energies of bMcS71L and bMcWT apo-b5 becomes larger with increasing [GdmCl]. Because holo-b5 stability is strongly dependent on apoprotein stability, this finding suggests that bMcS71L holo-b5 may actually be less stable than bMcWT b5 in the absence of denaturant. At the very least, the chemical denaturation data show that replacing Ser71 in bMcWT holo-b5 with Leu did not lead to a large increase in holoprotein stability.

The 1H NMR spectra of b5s exhibit paramagnetically shifted signals for several hemin substituent protons. Published assignments (Walker et al., 1988Go) for hemin substituent signals in both its more stable (A) and less stable (B) orientations in bMcWT b5 (the isomers are referred to hereafter as bMcWT,A and bMcWT,B b5, respectively) allowed ready assignment of many of the corresponding signals in the spectrum of the S71L mutant. 1H NMR spectra of bMcWT and bMcS71L b5 recorded after the ratio of hemin isomers had reached a constant value at 24°C show a complete reversal of hemin binding selectivity: from A:B {approx} 8:1 in bMcWT b5 to A:B {approx} 1:8 in bMcS71L b5 (Table III; Figure S2).


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Table III. Data from NMR experiments

 
For the purpose of examining the effect of the S71L mutation on the kinetic barrier for hemin release, bMcWT and bMcS71L apo-b5 were reconstituted with hemin at 4°C. As expected on the basis of previously reported studies (McLachlan et al., 1986Go; Walker et al., 1988Go), the 1H NMR spectra of each protein recorded at 4°C immediately after reconstitution showed that hemin orientations A and B were present in an ~1:1 ratio. No change in this ratio was observed to occur at 4°C over a 24 h period. Rate constants for conversion of the less stable (minor; m) to the more stable (major, M) orientation (km->M) were determined at 24°C in time-dependent NMR experiments using an established method (LaMar et al., 1981Go; Walker et al., 1988Go) (Figure 5; Table III).



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Fig. 5. Plots illustrating time-dependent changes in hemin isomer ratio for bMcWT b5 (A) and bMcS71L b5 (B) at 24°C. Lines through the data points represent fits to a monoexponential equation (see Table III).

 
From the equilibrium orientation ratio (Keq = [M]/[m] = km->M/kM->m), we calculated the corresponding rate constant for the reverse reaction (kM->m). The data show that the S71L mutation has modestly increased both km->M and kM->m.

We next compared rate constants for hemin transfer (k–H) from bMcWT and bMcS71L b5 to excess apoMb at 37°C and pH 7. Fits of kinetic curves for the reactions (Figure 6A; Table II) again show that bMcS71L b5 is more prone to hemin loss than is bMcWT b5. The hemin transfer studies were performed with samples containing the equilibrium ratio of A and B hemin orientations and k–H is therefore predominantly governed by loss of the major (more stable orientation) in each case (A in bMcWT b5; B in bMcS71L b5). The combined hemin transfer and hemin isomer equilibration data allow us to conclude that (1) hemin is released considerably more rapidly from bMcS71L,A b5 than from bMcWT,A b5 and even somewhat faster than from bMcWT,B b5; and (2) hemin is released more slowly from bMcS71L,B b5 than from bMcWT,B b5, but still somewhat faster than from bMcWT,A b5. In other words, replacing Ser71 in bMcWT b5 with Leu has lowered the kinetic barrier for release of hemin from orientation A, but has enhanced the kinetic barrier for release of hemin from orientation B to a similar extent. In this sense, our approach has been partially successful.



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Fig. 6. Comparison of hemin transfer data recorded at 37°C and pH 7. (A) FePPIX complexes of bMcWT, bMcS71L and bMcL32I/S71L b5. (B) FeDPIX complexes of bMcWT, bMcS71L and rOM b5.

 
In the light of the above discussion, it is noteworthy that the linewidths for all observable hemin substituent signals in bMcS71L,A b5 are substantially broadened in comparison with those for bMcS71L,B b5, as highlighted for the 5-methyl group (A5Me) in Figure 7A and Table III. In contrast, linewidths for hemin substituents in bMcS71L,B and bMcWT,B b5 are nearly identical, as shown for the 3-methyl group (B3Me) in Figure 7B and Table III. The data shown were recorded at 24°C, a temperature at which we have shown hemin orientational isomer equilibration to occur. The broadening of hemin signals in bMcS71L,A b5 relative to bMcS71L,B b5 is also present and equally significant in spectra recorded at 4°C, however, indicating that it is not a result of the exchange process itself. A common source of linewidth broadening in NMR spectra is interconversion between two or more conformational states with rate constants similar to the chemical shift difference (in Hz) for nuclei in those states (Sandström, 1982). The broadening of NMR signals for hemin in bMcS71L,A b5 relative to bMcWT,A b5 could therefore reflect enhanced mobility of the prosthetic group within its binding pocket and/or increased dynamic motion of the surrounding polypeptide. Either of these factors could be expected to compromise holoprotein stability, as we have observed. The linewidth data suggest at most a minor adverse effect of the S71L mutation on hemin and/or polypeptide dynamics in bMcWT,B b5. Hence, the increase in kinetic barrier for release of hemin from bMcWT,B b5 resulting from the S71L mutation indicated in hemin transfer and hemin isomer equilibration studies is probably not due to a decrease in frequency of conformational fluctuations that can trigger prosthetic group release. Indeed, linewidths for hemin substituents in bMcWT,B b5 are not particularly broadened relative to those for bMcWT,A, suggesting that the lower stability of the former is not due to adverse effects on core 1 dynamic properties. Rather, on the basis of our native-PAGE and DLS data, we propose that the increase in the kinetic barrier for release of hemin from bMcWT,B b5 resulting from the S71L mutation is due to a decrease in the extent of core 1 ‘opening’ that accompanies initial dissociation of hemin, which favors faster rebinding. This is another indication that our strategy for stabilizing bMcWT b5 has been at least partially successful. The same factor can be expected to mask the true impact of the S71L mutation on the kinetic barrier for release of hemin from bMcWT,A b5.



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Fig. 7. Signals for the A5Me (A) and B3Me (B) hemin substituents in NMR spectra of bMcWT b5 (top), bMcS71L b5 (middle) and bMcL32I/S71L b5 (bottom), normalized for peak height. Figure S2 (supporting information) compares the high-frequency regions of the original spectra, which were acquired at 24°C after the equilibrium ratio of hemin orientations had been reached.

 
The importance of steric complementarity between heme and its binding pocket

A major factor contributing to the large preference for hemin orientation A in bMcWT b5 is a hydrophobic grouping of amino acid side chains from ß4 (Leu23; Leu25) and ß5 (Ala54) (see Figure 2B). The side chains of these three amino acids create a steric environment accommodating the 3-methyl group of pyrrole ring II in bMcWT,A b5 (A3Me) better than the corresponding 2-vinyl group of pyrrole ring I in bMcWT,B b5 (B2V). The environment near the 2-vinyl group of pyrrole ring I in bMcWT,A b5 (A2V) is much less sterically congested. The hydrophobic side chain of Leu32 makes van der Waals contact with A2V from below (with the protein oriented as in Figure 2B), while the much smaller side chain of Ser71 makes contact with the same group from above.

The small preference for hemin orientation B relative to A exhibited by rOM b5 has been attributed to the presence of Leu at position 71 (see Figure 2C), which decreases the space available for a nearby A2V group in rOMA b5 (Altuve et al., 2001Go). Consistent with this argument, replacing Leu71 in rOM b5 with Ser has been shown to change the equilibrium hemin orientation ratio from A:B = 1:1.2 to 5.2:1 (Cowley et al., 2002Go). The complete reversal of the equilibrium hemin orientational ratio in bMcWT b5 resulting from the S71L mutation can therefore also be attributed to an increase in steric crowding that has a more adverse affect on the ability of the 2V group to fit comfortably (in bMcS71L,A) than it does on the smaller 3-methyl group when heme is present in orientation B (in bMcS71L,B).

As an initial approach for probing our hypothesis that steric strain between heme and its binding pocket contributes to destabilizing dynamic mobility in S71L bMc b5, we removed hemin (FePPIX) and replaced it with deuterohemin (FeIII deuteroporphyrin IX; FeDPIX). FeDPIX differs from FePPIX in having hydrogen atoms rather than vinyl groups at positions 2 and 4. Replacing FePPIX in wild-type bMc b5 with FeDPIX has previously been shown to cause a large increase in the rate at which hemin orientations A and B reach equilibrium starting from an ~1:1 ratio (McLachlan et al., 1986Go). Consistent with that report, our studies show that FeDPIX is lost from bMcWT b5 to apoMb 15-fold more rapidly than is FePPIX (Figure 6; Table II). The corresponding replacement in rOM b5 also lowered the barrier for prosthetic group release, but the extent of the effect could not be quantified. In contrast, the kinetic barrier for prosthetic group release from bMcS71L b5 increased when FePPIX was replaced by FeDPIX. In fact, the rate constant for prosthetic group loss from the FeDPIX complex of bMcS71L b5 is 16-fold smaller than that for the FeDPIX complex of bMcWT b5 and only about 2-fold greater than that exhibited by the FeDPIX complex of rOM b5.

Identification of the most likely source of steric strain in the FePPIX complex of bMcS71L b5 was informed by our previous studies in which residues contributing to conserved hydrophobic packing motifs a and b in rOM b5 were replaced with the corresponding bMc b5 residues (Altuve et al., 2001Go; Cowley et al., 2002Go). Replacing Ile25 in motif b of rOMWT b5 with Leu barely altered the A:B ratio, nor did it significantly affect hemin release kinetics (Cowley et al., 2002Go). In contrast, replacing Ile32 in motif a of rOMWT b5 with Leu led to an increase in relative stability of orientation A (from A:B = 1:1.2 to 4:1) and markedly accelerated both hemin orientational isomer equilibration and hemin transfer (Altuve et al., 2001Go). This can be attributed to the fact, noted above, that the side chains of residues 71 and 32 impinge upon the 2-vinyl group from above and below the mean plane of the prosthetic group in orientation A. The presence of two {delta}-methyl groups on Leu as opposed to one in Ile significantly increases steric crowding near the A2V group when residue 71 is also a Leu.

To probe the hypothesis that the presence of Leu at positions 71 and 32 is a major source of steric strain in bMcS71L b5, we generated the L32I/S71L double mutant (bMcL32I/S71L b5). The S71L and L32I/S71L mutants exhibit similar unfolding free energies at 25°C as determined in GdmCl-mediated denaturation studies (Figure S1A; Table II), consistent with the conservative nature of the second mutation. However, the L32I mutation significantly increases the holoprotein Cm value (Figure S1B; Table II), rendering it much more OM b5-like and suggesting an increase in the kinetic barrier for hemin release. The latter was verified in hemin transfer studies performed at 37°C and pH 7, which show that bMcL32I/S71L b5 loses hemin ~5-fold more slowly than does bMcS71L b5 and 3-fold more slowly than does bMcWT b5 (Figure 6A; Table II). The effect is considerably smaller than observed upon replacement of FePPIX with FeDPIX in bMcWT and bMcS71L b5, but nonetheless suggests that we correctly identified a key source of the steric strain between hemin and the polypeptide in the S71L mutant.

Table III shows that introducing the L32I mutation into bMcS71L b5 substantially increases the population of hemin orientation A relative to orientation B as determined by NMR, from A:B = 1:8 (Figure S2B) to 1.4:1 (Figure S2C). In fact, the equilibrium ratio of hemin orientations in bMcL32I/S71L b5 is very close to that exhibited by rOM b5 (A:B = 1:1.2; Figure S2D). In addition, the linewidths for hemin isomer A substituents in bMcL32I/S71L b5 are greatly narrowed in comparison with their counterparts in the spectrum of bMcS71L b5 (Figure 7A; Table III). We interpret these findings as indicating that the L32I mutation has primarily increased the hemin release kinetic barrier in bMcS71L,A b5, by damping dynamic motion in the heme binding pocket. It is noteworthy that the A5Me linewidth for bMcL32I/S71L b5 is narrower than the corresponding signal in bMcWT b5, but remains broad in comparison with the signal for A5Me in the spectrum of rOM b5. Hence the stability and linewidth studies agree in indicating that bMcL32I/S71L b5 exhibits stability and dynamic properties midway between those of bMcWT and rOM b5. We tentatively conclude that heme and the surrounding polypeptide in core 2 of bMcL32I/S71L b5 still suffers from steric strain in comparison with rOM b5, allowing more frequent, albeit less extensive, conformational changes that can trigger release of the prosthetic group.

Conclusion

Our studies suggest that heme-binding pocket compactness and dynamic mobility in the apo state of b5 can be varied over a wide range by shifting the balance of hydrophobic and hydrophilic residues in core 1. As demonstrated most convincingly by the FeDPIX complexes of bMcWT and bMcS71L bMc b5, increased binding pocket compactness can substantially retard the rate at which hemin is released and thereby enhance holoprotein thermodynamic stability. Hence we believe that our studies provide new general principles that can be applied toward the design of b-heme proteins exhibiting improved stability properties, perhaps for use in biotechnological applications. Our strategy of enhancing Mc b5 stability via reduction in core 1 size and dynamic mobility in the apo state has its roots in the preorganization principle enunciated by Cram (Cram, 1988Go) and complements previously published approaches that focus on the holo state including (1) introduction of a disulfide linkage that bridges cores 1 and 2 (Storch et al., 1999aGo,bGo); (2) engineering a covalent linkage between heme and the polypeptide (Barker et al., 1993Go; Lin et al., 2005Go); and (3) improving core 1 secondary structure stability in the holo state by replacing residues in helices with others having greater helical propensity or by relieving electrostatic repulsion (Mukhopadhyay and Lecomte, 2004). Our strategy nonetheless requires attention to the holo state in order to avoid introducing steric bulk that prevents heme from seating comfortably in its binding pocket. Finally, our studies suggest that hemin substituent linewidths are sensitive indicators of steric strain. Hence, comparison of linewidths can nicely complement hemin transfer and hemin orientational equilibration data as approaches for quickly assessing effects of mutations on heme binding properties. MD simulations and HDX experiments monitored by NMR spectroscopy will allow us to investigate the detailed structural basis for interesting cases and to further our understanding of the relationship between dynamics and stability in heme proteins and their possible involvement in function.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work was supported by a grant from the National Science Foundation (MCB-0446326). We thank Professor Russell Middaugh for use of his DLS apparatus.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
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Received May 27, 2005; revised July 26, 2005; accepted September 1, 2005.

Edited by Michael Hecht





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