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1ß1, 1ß2, and 12 interfaces
1 Department of Biochemistry, College of Medicine, The University of Iowa, Iowa City, Iowa 52242, USA
2 Department of Medicine and Biochemistry, School of Medicine, Veterans Administration Medical Center, State University of New York, Buffalo, New York 14215, USA
3 Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy
4 Italian National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy
Reprint requests to: Arthur Arnone, Department of Biochemistry, College of Medicine, The University of Iowa, Iowa City, Iowa 52242, USA; e-mail: arthur-arnone{at}uiowa.edu; fax: (319) 335-9570.
(RECEIVED May 2, 2001; FINAL REVISION June 14, 2001; ACCEPTED June 14, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/
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Abstract |
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1ß1, 1ß2, and 12 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins ßY35F and ßY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35ß results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in ßY35F and increases in ßY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in ßY35F or ßY35A. X-ray crystal structures of deoxy ßY35F and ßY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The ßY35F mutation repositions the carboxyl group of Asp1261 so that it may form a more favorable interaction with the guanidinium group of Arg1412. The ßY35A mutation results in increased mobility of the Arg141 side chain, implying that the interactions between Asp1261 and Arg1412 are weakened. Therefore, the changes in the functional properties of these 35ß mutants appear to correlate with subtle structural differences at the C terminus of the -subunit. Keywords: Hemoglobin; mutant; ligand-binding kinetics; ligand affinity; X-ray crystallography; single-crystal microspectrophotometry
Abbreviations: Bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane • Tris, tris(hydroxymethyl)aminomethane • EDTA, disodium ethylenediaminetetraacetate • PEG, polyethylene glycol • r.m.s., root mean square • IHP, inositol hexaphosphate • HbA, human hemoglobin major component • ßV1M, recombinant hemoglobin with the Val 1ß 
Met mutation • ßY35F, recombinant hemoglobin with the Tyr 35ß Phe and Val 1ß Met mutations • ßY35A, recombinant hemoglobin with the Tyr 35ß Ala and Val 1ß Met mutations • met-hemoglobin, hemoglobin with iron oxidized to Fe(III).
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Introduction |
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2ß2 hemoglobin tetramer generates an - interface (designated 12), a ß-ß interface (designated ß1ß2), and two types of -ß interfaces (designated 1ß1 and 1ß2). The 12 and ß1ß2 interfaces form a solvent channel that is almost devoid of direct intersubunit contacts and provides binding sites for a variety of allosteric effectors (Perutz 1989; Perutz et al. 1994). The only direct contacts between like subunits occur at the 12 interface, where ionic interactions form between the C-terminal residue of one -subunit, Arg141, and residues Asp126 and Lys127 on the opposite -subunit. In contrast, extensive contacts are formed between unlike subunits at the 1ß1 and 1ß2 interfaces. These two interfaces, however, have very different characteristics. The 1ß2 interface is highly polar and dynamic, undergoing large changes in structure as a function of ligation state, whereas the 1ß1 interface is static and less polar. The most direct way to determine the relative importance of residues that contribute interactions to the subunit-subunit interfaces of the hemoglobin tetramer is to perform detailed site-directed mutagenesis studies on these residues. We did this in the case of residue 35ß by creating and performing complementary structural and functional studies on the mutants ßY35F and ßY35A.
Located at the convergence of the 1ß1, 1ß2, and 12 interfaces, Tyr35ß contributes to a complex network of atomic interactions through which it can potentially influence the structure and stability of each of these interfaces (see Fig. 1
). In the case of the 1ß1 interface, the phenolic hydroxyl group of Tyr35ß1 makes two intersubunit interactions, one direct and one indirect. The direct interaction consists of a hydrogen bond to the side chain carboxylate oxygen, O
2, of Asp1261. The indirect interaction consists of an intersubunit water bridge between Tyr35ß1 and the side chain of His1221 (water molecule W2 in Fig. 1). The hydroxyl group also interacts with another water molecule, W1, that is part of a network of water molecules (W1 through W6 in Fig. 1) at the 1ß1 interface. In the case of the 2ß1 interface (or the symmetry-related 2ß1 interface), the side chain of Tyr35ß1 makes van der Waals contacts between the edge of its phenolic ring and the guanidinium side chain of Arg1412. In the case of the 12 interface, Tyr35ß1 interacts with both Asp1261 and Arg1412, and therefore, it can potentially influence the structure and stability of this interface as well. Additionally, Tyr35ß1 can indirectly influence the 12 interface through van der Waals contacts it makes with Val34ß1, because Val34ß1 makes van der Waals contacts with Asp126ß1 and a hydrogen bond with Arg1412. Tyr35ß1 and Val34ß1 are unique in that they are the only residues that contribute atomic interactions to both the 1ß1 and 1ß2 interfaces.
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Results |
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. Under these conditions, the combination of CO with human hemoglobin A is an autocatalytic process that, when fitted to a single exponential function, has an overall rate constant that varies from 0.09 x 106 M-1sec-1 to 0.31 x 106 M-1sec-1 depending on the pH and presence or absence of IHP. At pH 6 and 7, the ßY35F substitution slows these reactions slightly without affecting their autocatalytic nature. At pH 8, the kinetic difference between native hemoglobin A and ßY35F increases because of a decreased pH dependence of the kinetic properties of the mutant.
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CO recombination following photodissociation
Kinetics of CO recombination following flash photolysis are typically heterogeneous with two kinetic phases, the rates of which differ by more than an order of magnitude. The slower of the two kinetic phases is associated with a rate constant similar to that measured for the combination of CO with the deoxygenated tetramer following rapid mixing. For ßY35F and ßY35A, rate constants for the slow kinetic phases (Table 2) agree well with those measured under the same conditions by rapid mixing in the stopped flow apparatus (Table 1
). The rapid phase is caused by ß dimers. The magnitude of this phase is concentration dependent and varies with the presence and concentration of organic phosphates such as IHP. At a concentration of 100 µM IHP, the relative amount of dimers is reduced as a result of preferential binding of IHP to the ligand-saturated hemoglobin tetramer. This pattern is observed in Table 2 for the CO recombination kinetics of wild-type hemoglobin and the two 35ß variants at pH 7 in the presence and absence of IHP. The ratio of the magnitudes of the fast and slow kinetic phases offers a measure of the extent to which the ligand-saturated hemoglobin tetramer dissociates into ß dimers. In this regard, the two variants appear very similar to one another but are slightly more dissociated than wild-type hemoglobin. The data in Table 2 predict values of -7.8 ± 0.1 kcal/mole for HbA, in agreement with the results of Doyle et al. (1992); -7.4 ± 0.1 kcal/mole for ßY35F; and -7.3 ± 0.1 kcal/mole for ßY35A. A value of -7.7 ± 0.2 kcal/mole determined by the same method was previously reported for ßV1M by Doyle et al. (1992).
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. The equilibrium-binding properties are summarized in Table 3. If p50 is taken as an approximation of p-median for the calculation of average free energy of oxygen binding, then the binding of oxygen is made 3.3 kcal/4 mole O2 more favorable by the ßY35A substitution and 1.2 kcal/4 mole O2 less favorable by the ßY35F substitution. The cooperativity (n-Hill in Table 3) is unchanged by the ßY35A mutation. In the case of the ßY35F mutation, the Hill coefficient is decreased slightly by 0.4, but this may not be significant because the standard deviation of the difference in n is 0.28 (= 0.2 x [2]1/2 based on the data in Table 3).
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The relationships between changes in the free energy of oxygen binding and changes in the free energy of dimer assembly can be analyzed by means of the linkage scheme:
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G2 for ßY35F and ßY35A are only slightly increased compared with HbA and ßV1M. The comparison of fully liganded CO and oxyhemoglobins is meaningful, because at pH 7.4 these liganded forms have the same free energy of assembly (Huang and Ackers 1996). The assumption that the oxygen affinity of the ß dimers is not affected by the modification of 35ß is supported by the similarities of the rate constants associated with the rapid phases of the recombination kinetics and by the location of this residue. If this is accepted, then there is a direct relationship between the oxygen affinity of the tetramer and the free energy of association of the deoxygenated ß dimers. The slight reduction in the oxygen affinity of ßY35F indicates little change in the stability of the deoxygenated tetramer of this variant. This is inconsistent with the strong destabilization of the deoxy tetrameric structure in the micromolar range of heme concentration that would be expected from the previous Hb Philly studies (Rieder et al. 1969; Asakura et al. 1976, 1981). This indicates that the reported Hb Philly mutation (Tyr35ß Phe) is incorrect. The same conclusion was reached by Nakatsukasa et al. (1998) as a result of their examination of an independently prepared ßY35F variant of HbA. On the other hand, the increased oxygen affinity of ßY35A in conjunction with its relatively unchanged 4G2 requires a significant destabilization of its deoxygenated tetramer, i.e., an increase in 0G2. This destabilization of the deoxygenated ßY35A is evidenced by the hetergeneity of CO combination at pH 8 in the absence of IHP, which is probably caused by the presence of rapidly reacting deoxygenated ß dimers.
Equilibrium measurements of oxygen binding to crystals of quaternary-T hemoglobin
Crystals of ßY35F and ßY35A were grown anaerobically from polyethylene glycol (PEG) solutions. Crystals of deoxyhemoglobin which grow from PEG solutions are orthorhombic with the b crystal axis usually having the shortest dimension. It is for this reason that the spectra are normally taken only with the light polarized parallel to the a or c crystal axes. This also was found to be the case for crystals of ßY35F. However, all of the crystals of ßY35A examined had grown with their shortest dimension along the a crystal axis. For this variant, spectra were obtained with light polarized parallel to the b or c crystal axes.
Polarized absorption spectra for ßY35F and ßY35A crystals were determined at several oxygen pressures. Hill plots of the oxygen-binding data are shown in Figure 3. In Table 4, the p50s and Hill coefficients obtained from the data in Figure 3 are compared with the same parameters for wild-type hemoglobin. The Hill coefficients for the mutant and wild-type hemoglobins are close to 1 because the crystal lattice restricts the quaternary structure to T-like conformations. The oxygen affinity and Hill coefficient of crystalline ßY35F are slightly lower than those of crystalline hemoglobin. Measurements of the fractional saturation of ßY35F crystals at a single oxygen pressure as a function of pH indicate no significant Bohr effect (data not shown). The lack of a Bohr effect in crystalline ßY35F is consistent with previous studies on wild-type hemoglobin crystals (Rivetti et al. 1993a). As can be seen in panel b of Figure 3, data were obtained on the ßY35A variant only up to 30% oxygen saturation. Above this level of oxygenation, ßY35A crystals become unstable and show irreversible increases in oxygen affinity. Again, as seen in Table 4, both the oxygen affinity and Hill coefficient are greater for ßY35A crystals than for crystals of HbA. The unusual instability of these crystals with respect to oxygenation may contribute to the increased Hill coefficients.
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1 and the hydroxyl group of Tyr35ß1 leads to a small shift in the benzene ring of Phe35ß1, a slight rotation (
15°) of the Asp1261 carboxyl group, and small changes in the associated water molecule network at the 1ß1 interface (Fig. 4). In particular, water W4 shifts by 0.8 Å to maintain its interaction with Asp1261, and the mobility of water molecule W1 increases (as indicated by a 65% increase in its temperature factor), reflecting the loss of its interaction with the hydroxyl group of Tyr35ß1. In the case of ßY35A, removal of the entire phenolic ring eliminates van der Waals contacts with Thr34ß1, Leu105ß1, and Arg1412 as well as the polar interactions associated with the OH group. Creation of the large cavity in ßY35A leads to increases in the atomic temperature factors of residues Thr34ß1, Leu105ß1, and Arg1412 (Fig. 5) and the elimination of water molecular W1, but it does not result in significant atomic displacements of the adjacent residues (Fig. 4). It is important to note that although the structural changes associated with mutations ßY35F and ßY35A are very small, virtually identical mutation-induced changes are associated with both residue 35ß1 and residue 35ß2 (data not shown).
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Discussion |
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1ß1, 1ß2, and 12 interfaces in the deoxyhemoglobin tetramer. In fact, hemoglobin Philly (Rieder et al. 1969; Asakura et al. 1976, 1981), a naturally occurring mutant hemoglobin with very high oxygen affinity, decreased cooperativity, and decreased stability, was reported to be a Tyr35ßPhe substitution. Moreover, it was indicated, based on the increased sulphydryl reactivities of cysteines 104 and 112ß, that the mutation in hemoglobin Philly destabilized the 1ß1 interface, shifting the monomer dimer equilibrium toward monomer formation. However, Nakatsukasa et al. (1998) performed oxygen equilibrium measurements in solution on the recombinant ßY35F variant and found this mutant hemoglobin to have a slightly increased p50 and normal cooperativity. Therefore, they concluded that the hemoglobin Philly mutation must be something other than a Tyr35ßPhe substitution. Nagai et al. (1999) further reported that the difference ultraviolet resonance Raman spectrum between the deoxy and CO forms of ßY35F is nearly identical to that of HbA, supporting this conclusion. In agreement with these results, we find that the kinetics of CO combination of ßY35F in solution and the oxygen affinity of crystalline ßY35F indicate that the deoxy quaternary-T structure of this mutant has slightly reduced ligand affinity and is not destabilized significantly. In addition, the CO recombination kinetic data reported above indicate that the ßY35F mutation causes little, if any, difference in the stability of the fully oxygenated tetramer.
The fact that the ßY35F mutation does not significantly reduce the stability of the deoxy tetramer implies that the Tyr35ß1• • • • Asp1261 hydrogen bond makes no significant contribution to the stability of the deoxy HbA tetramer, or alternatively that loss of this interaction in ßY35F is compensated for by other interactions. If the former possibility is true, the Tyr35ß1• • • • Asp1261 hydrogen bond may still be present in isolated ß dimers, or the interaction of water with Tyr35ß and Asp126 is energetically equivalent to the Tyr35ß1• • • • Asp1261 hydrogen bond. If the latter is true, crystallographic analysis of the deoxy ßY35F structure indicates a stronger interaction between Asp1261 and Arg1412 may compensate for the loss of the Tyr35ß1• • • • Asp1261 hydrogen bond. In the wild-type deoxyhemoglobin tetramer, the side chain of Asp1261 is buried between the 1-, ß1-, and 2-subunits, where its O2 atom makes three intersubunit interactions with other buried polar atoms. Specifically, the O2 atom interacts with the hyroxyl group of Tyr35ß1 and the N
1 and N2 atoms of the Arg1412 guanidinium group. In deoxy ßY35F, however, the O2 atom of Asp1261 only interacts with Arg1412 because the 35ß hydroxyl has been eliminated. Consequently, the interactions between the O2 atom and the Arg1412 guanidinium group should, in principle, have increased strength in the ßY35F mutant. The rotation of Asp1261 carboxyl group observed in deoxy ßY35F crystal structure (Fig. 4) is consistent with this possibility, because the rotation repositions the O2 atom so that it points more directly at the Arg1412 guanidinium group. It has been well documented that the stability of the deoxy hemoglobin tetramer is strongly linked to the 12 interactions associated with Arg141 (Antonini et al. 1961; Perutz 1970; Perutz and TenEyck 1972; Bonaventura et al. 1974; Kilmartin et al. 1975; Kavanaugh et al. 1995). Therefore, slightly stronger interactions between Asp1261 and Arg1412 may be the stereochemical basis for the slightly reduced ligand affinity and cooperativity of the quaternary-T ßY35F (as observed in crystals of ßY35F) as well as the overall decreases measured in solution.
Although the loss of the hydrogen bond associated with Tyr35ß has a very small stabilizing effect on the deoxyhemoglobin tetramer, the loss of the van der Waals interactions associated with the benzene ring of Tyr35ß destabilizes the deoxy tetramer and increases the oxygen affinity of the quaternary-T structure. The oxygen-binding isotherm measured in solution (Fig. 2) clearly shows that ßY35A has increased oxygen affinity. Collectively, the oxygen-binding measurements made on crystals (Fig. 3) and the CO combination (Table 1) and recombination rates (Table 2) measured in solution, indicate that this increase in ligand affinity is in large measure because of changes in the deoxy tetramer. Because the magnitudes of the fast and slow CO-rebinding phases following flash photolysis are very similar for ßY35A and HbA (Table 2), it can be concluded that the ßY35A mutation has little effect on the stability of the liganded structure. The faster CO combination rates measured in the presence of IHP (Table 2) and lower p50 measured for ßY35A crystals (Table 3) both indicate that the quaternary-T structure of the ßY35A tetramer has increased oxygen affinity.
The increased oxygen affinity and decreased stability of the deoxy ßY35A tetramer correlate with increased mobility of the Arg141 in the deoxy ßY35A crystal structure (Fig. 5). A similar observation has been made for a series of site-directed mutants at position 37ß (Kavanaugh et al. 1992b, 1998; ; Rivetti et al. 1993b; Kelly et al. 1994; Kiger et al. 1998; Kwiatkowski et al. 1998; Peterson and Friedman 1998) and for deoxy desArg hemoglobin (Kavanaugh et al. 1995). Presumably the increased mobility at the -subunit C termini in the ßY35A, desArg hemoglobin, and the 37ß mutant deoxyhemoglobin structures results in increased oxygen affinity by allowing ligation-induced changes in structure to be more easily accommodated.
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Materials and methods |
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-globin as described by Hernan et al. (1992). Approximately 110 mg of ßY35F hemoglobin and 210 mg ßY35A hemoglobin were obtained from 24 L of bacterial culture.
Measurement of CO combination kinetics by rapid mixing
Rapid-mixing CO combination kinetics were performed with an OLIS (On Line Instrument Systems Inc.) stopped-flow apparatus, which is similar to the one first described by Gibson and Milnes (1964). The procedure for measuring the rate of CO combination with deoxygenated hemoglobin was similar to that of Gibson (1959). The time course of the reaction was followed by monitoring the change in absorbance at 420 nm and 435 nm using a cuvette with a 1.7-cm pathlength. Concentrations of CO and hemoglobin (in heme equivalents) were 20 µM and 2 µM, respectively. Dithionite was present at a concentration of 2 mM.
Measurements of CO recombination following photodissociation
Measurements of CO recombination following photodissociation were performed as previously described (Doyle et al. 1992). Photolysis was by means of the simultaneous discharge of three photographic strobe units (Sunpak Auto 544) equipped with thyristor quenching devices. These were adjusted to produce a rectangular pulse of light 0.5 msec in duration. The flash radiation was filtered through solutions of auramine, which has high extinction coefficients at 420 nm and 435 nm, the wavelengths at which the time course was monitored. This reduced or eliminated interference between the flash and the measurement of changes in absorbance as a function of time. Reactant concentrations were the same as those used in the rapid-mixing experiments.
Measurements of oxygen-binding equilibrium in solution
Oxygen-binding curves were measured by tonometry essentially by the Nagel et al. (1965) modification of the method of Allen et al. (1950). A 500-mL tonometer with an attached 2-mm pathlength cuvette was used. The Hb concentration was 160 µM in heme equivalents. Spectral measurements were performed with a Cary 14 spectrophotometer modified by OLIS for computer control and online data acquisition. Measurements were performed at 20°C in 100 mM HCl–bis-Tris (HCl–bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane) buffer at pH 7. This buffer was prepared by titrating HCl against the base form of bis-Tris and then diluting the resulting solution with deionized water to a final chloride concentration of 0.1 M. Deoxygenation was accomplished by equilibration with oxygen-free nitrogen. Reduction system enzymes (Hayashi et al. 1973) were added to prevent metHb formation.
Microspectrophotometric measurements of oxygen binding to hemoglobin crystals
The small (100 to 150 µm long and 20 µm thick) crystals required for microspectrophotometry were grown from PEG 8000 (Hampton Research) solutions at room temperature as previously described (Rivetti et al. 1993b). Once grown, the crystals were washed first with 20% (w/v) and then 36% (w/v) anaerobic PEG containing 10 mM phosphate at pH 7.2 and 30 mM sodium dithionite. Crystals were then stored at 4°C until used for data collection.
Before spectra were collected, single crystals of either ßY35F or ßY35A were first resuspended in 36% (w/v) PEG 8000, 10 mM potassium phosphate, 1 mM EDTA, 30 mM dithionite at pH 7.0. Then crystals of ßY35F were suspended eight times in a solution containing 54% (w/v) PEG 8000, 10 mM potassium phosphate, 3270 U/ml catalase at pH 7.0, unless otherwise stated, and loaded into the Dvorak-Stotler flow cell (Dvorak and Stotler 1971) in air. Crystals of ßY35A crack on exposure to air; therefore, the PEG solution was saturated with nitrogen, and the resuspension and loading into the Dvorak-Stotler cell took place in an anaerobic glove box.
Polarized absorption spectra were recorded with the electric vector of the linearly polarized light parallel to the a and c optical axes for crystals of ßY35F and parallel to the b and c axes for crystals of ßY35A. Oxygen pressures between 0 and 760 torr were obtained from gas mixtures prepared using a gas-mixing generator, Environics 200. Oxygen-binding curves were determined at 15°C. The fractional saturation with oxygen and the fractional concentration of oxidized hemes were calculated by fitting of the observed spectra to a linear combination of reference spectra from deoxy, oxy, and oxidized hemoglobin crystals plus baseline (Rivetti et al. 1993a). In the case of ßY35F crystals, HbA reference spectra were used, whereas in the case of ßY35A crystals, reference spectra were obtained by exposing ßY35A crystals to a dithionite-containing solution (deoxyhemoglobin spectra), then after removal of dithionite, to a solution containing 5 mM ferricyanide (oxidized reference spectra). Oxyhemoglobin reference spectra were obtained by exposing crystals to an oxygen pressure of 760 torr at 5°C. Under these conditions, the crystals are fully saturated and still birefringent, despite evident crystal cracks. All measurements were performed with a content of oxidized hemes <15%.
X-ray diffraction analysis
Before crystallization, all mutant hemoglobins were stripped of organic and inorganic ions by passing them over a Dintzis column (Riggs 1981), which was modified by the addition of a 1-mm layer of chelating resin (iminodiacetic acid, Sigma #C-7901) to the top of the column. The stripped oxyhemoglobins were frozen and stored in liquid nitrogen until used for crystallization.
Monoclinic crystals (space group P21) of the mutant deoxyhemoglobins were grown in 100-µL batch set-ups at room temperature from solutions of concentrated ammonium sulfate, as described by Perutz (1968) for deoxyhemoglobin A, except that 10 mM ferrous citrate was used as the reducing agent. Crystals of ßY35F (unit cell dimensions: a = 63.2 Å, b = 83.7 Å, c = 53.8 Å, and ß = 99.4°) and ßY35A (unit cell dimensions: a = 63.2 Å, b = 83.7 Å, c = 53.7 Å, and ß = 99.3°) were isomorphous with ßV1M crystals (unit cell dimensions: a = 63.2 Å, b = 83.7 Å, c = 53.8 Å, and ß = 99.4°). All crystallization solutions were thoroughly deoxygenated before their use, and all crystallization work was conducted in a glove bag that was continuously purged with nitrogen.
Single crystals of the ßY35F and ßY35A mutants were washed briefly in crystallization buffer in which the total salt concentration was increased to 2.6 M (Perutz 1968) and then mounted in quartz capillaries for data collection. Diffraction data were collected on a Rigaku AFC6 diffractometer fitted with a San Diego Multiwire Systems area detector. All diffraction data were scaled and merged according to the procedure of Howard et al. (1985). In each case, degradation because of radiation damage was <15% as determined from a subset of diffraction data that was collected at the beginning and end of data collection. Data were collected out to a resolution of 1.8 Å (48,043 independent reflections) and 1.7 Å (55,581 independent reflections), respectively, for the crystals of the ßY35A and ßY35F mutants. The Rsymm values for the ßY35A and ßY35F data sets are 6.1% and 6.3%, respectively. Each mutant data set was split into a working set, consisting of 90% of the data, that was used for refinement, and a test set, consisting of 10% of the data, that was used to calculate Rfree (Brünger 1992a).
Refinement of the deoxy ßY35F and ßY35A crystal structures consisted of rigid body refinement using X-PLOR (Brünger 1992b) followed by restrained least-squares refinement with PROLSQ (Hendrickson 1985; Sheriff 1987). The initial atomic model for both refinements was the 1.8 Å structure of deoxyhemoglobin ßV1M (Kavanaugh et al. 1993) in which Tyr35ß was converted to a phenylalanine or an alanine. The standard crystallographic R values for the initial ßY35F and ßY35A models were 0.177 and 0.174, respectively, for data between 8.0 Å and 1.8 Å resolution with magnitudes >2
(48,021 reflections for ßY35F and 42,698 reflections for ßY35A). Twenty cycles of rigid body refinement of the entire tetramer, followed by 20 refinement cycles of the two ß dimers, and finally 20 refinement cycles of the four individual subunits resulted in an R value of 0.170 for both mutant structures. Following rigid body refinement, the structures were subjected to 15 cycles of restrained least-squares refinement, which included refinement of individual atomic temperature factors. The resulting ßY35F and ßY35A atomic models have R values of 0.162 and 0.157, respectively, and corresponding Rfree values of 0.218 and 0.224. The final atomic models have excellent stereochemistry with root mean square (rms) deviations from ideal bond lengths of 0.014 Å and 0.016 Å and rms deviations from ideal angles of 1.7° and 1.6° for ßY35F and ßY35A, respectively. The mutant atomic models were analyzed using the least-squares superposition methods previously described (Kavanaugh et al. 1992b, 1998).
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References |
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