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• W2114656295 abstract "The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K.(1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity ∼5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature. The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K.(1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity ∼5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature. INTRODUCTIONMyoglobin is a small, 16-18-kDa, globular hemeprotein which stores molecular oxygen in muscle cells. Because of the similarity of their tertiary structures, myoglobin is frequently used as a model for the α and β subunits of tetrameric hemoglobin. The key physiological properties of myoglobin are its high affinity for oxygen and low rate of spontaneous oxidation to the inactive ferric form (Antonini and Brunori, 1971). Studies of naturally occurring and mutant hemeproteins have shown that ligand affinity and autooxidation are affected markedly by the chemical nature of the amino acids in the heme pocket. In order to understand these functional properties, the molecular structures of a large number of myoglobins and hemoglobins have been determined by x-ray crystallography and solution NMR studies. These studies have been pursued along two lines. Traditionally, naturally occurring genetic or species variants with distinctive functional properties have been investigated (i.e. Bolognesi et al.(1989), Steigmann and Weber(1979), Vainshtein et al.(1978), Yu et al.(1990), Qin and La Mar (1992), and Vyas et al.(1993)). More recently, site-directed mutagenesis has been used either to test structural hypotheses (Olson et al., 1988; Braunstein et al., 1988; Carver et al., 1990; Ikeda-Saito et al., 1991; Balasubramanian et al., 1993; Li et al., 1994; Ling et al., 1994; Huang and Boxer, 1994) or to explore systematically the physiological roles of individual residues (Rohlfs et al., 1990; Egeberg et al., 1990; Smerdon et al., 1991, 1993; Carver et al., 1991, 1992; Quillin et al., 1993, 1995; Lai et al., 1995). A review of the mutagenesis literature is given in Springer et al.(1994).Dene et al.(1980) sequenced Asian elephant myoglobin and reported that it contains a Gln at position 64 (E7) rather than a His, which is found at this position in all other mammalian myoglobins. This replacement appeared to be neutral since the O2 affinity and autooxidation rate of elephant myoglobin are very similar to those of pig, human, and, sperm whale Mb 1The abbreviations used are: MbdeoxymyoglobinMbCOcarbonmonoxy myoglobinNOESYtwo-dimensional nuclear Overhauser effect spectroscopyCOSYtwo-dimensional bond correlation spectroscopyMCOSYmagnitude COSYTOCSYtwo-dimensional total correlation spectroscopyWEFTwater-eliminated Fourier transform. (Table 1, Romero-Herrera et al., 1981; Springer et al., 1994). Romero-Herrera et al.(1981) concluded that in elephant myoglobin, N∊ of Gln64(E7) forms a hydrogen bond with bound oxygen which is equivalent in strength to that formed with His64(E7) in all other mammalian myoglobins. However, when this idea was tested directly by constructing the His(E7) → Gln mutation in recombinant sperm whale myoglobin, the results indicated that this substitution is not neutral. The H64Q mutation causes a 5-fold decrease in KO2Tabled 1 Open table in a new tab Studies aimed at understanding why H64Q sperm whale myoglobin does not mimic the functional and spectral properties of elephant myoglobin have been pursued by two approaches. First, La Mar and co-workers (Yu et al., 1990; Vyas et al., 1993) have carried out high resolution 1H NMR studies of both diamagnetic carbonyl and paramagnetic cyano-met complexes of elephant Mb and compared the results to native sperm whale myoglobin as a standard. The spectra show that, in addition to features for the expected distal Gln64(E7), there are also signals which strongly indicate that a phenylalanine side chain is in van der Waals contact with bound ligand in the elephant protein.Second, Olson, Phillips, and co-workers (Carver et al., 1992; Gibson et al., 1992; Springer et al., 1994; Li et al., 1994) have carried out a systematic study of point mutations at position 29 (B10) in sperm whale myoglobin. Substitution of Phe for Leu29(B10) in sperm whale myoglobin leads to a dramatic increase in O2 affinity and marked decrease in autooxidation rate (Carver et al., 1992). The crystal structure of this mutant shows that Cζ of the Phe29 side chain makes van der Waals contact with bound ligands. The Feδ(+)-O-Oδ(−) complex is stabilized by favorable electrostatic interaction with the positive edge of the phenyl multipole, accounting partly for the 15-fold increase in affinity shown by the L29F mutant. The large Phe29 side chain also excludes water from the distal pocket of the deoxy form of the mutant. 2The structure of L29F deoxymyoglobin has been determined to 1.7 Å by M. L. Quillin as a part of his Ph.D. thesis work. The orientations of the His64(E7) and Phe29(B10) side chains are very similar to those reported for the met, CO, and oxy structures of this mutant. A novel feature of the L29F deoxymyoglobin structure is the absence of an internal, distal pocket water molecule due to the large size of the Phe29 residue. In native and wild-type sperm whale deoxymyoglobin, a non-coordinated water molecule is found hydrogen-bonded to N∊ of His64(E7) and restricts access to the heme iron atom (Quillin et al., 1993). The favorable multipole and water exclusion effects also inhibit protonation of the Feδ(+)-O-Oδ(−) complex, preventing disproportionation into Fe3+ and HO2· (Brantley et al., 1993).In an effort to construct a blood substitute prototype, Olson and co-workers (Brantley et al., 1993; Olson, 1994) constructed a double mutant in which Phe was put at the 29 position to inhibit autooxidation by excluding water from the active site and Gln was put at the 64 position to lower O2 affinity by weakening hydrogen bonding to the bound ligand. Later, it was discovered that this active site configuration, Phe29(B10)/Gln64(E7), was predicted to occur in elephant Mb by Yu et al.(1990) even though the published sequence for the elephant protein lists a Leu at the B10 position (Dene et al., 1980). To resolve this problem and test the conclusions of Yu et al.(1990), we have compared in detail the structural, spectral, and functional properties of elephant myoglobin and the L29F/H64Q sperm whale mutant. Rate constants for O2 and CO binding and autooxidation were determined by conventional kinetic techniques. Infrared spectra of the CO complexes of wild-type, L29F, H64Q, and L29F/H64Q sperm whale myoglobins were compared with that of elephant MbCO. Resonance Raman techniques were used to measure the νFe-CO and νFe-CN bands for the same set of mutants and native proteins. The structure of the active site of the cyano-met form of the sperm whale double mutant was determined by solution 1H NMR and compared with that of the single mutants and elephant myoglobin. Finally, the structure of L29F/H64Q sperm whale MbCO was determined by x-ray crystallography for comparison with the active site parameters determined by NMR and with the crystal structure of native elephant metmyoglobin recently obtained by Bisig et al.(1995).MATERIALS and METHODSSite-directed Mutagenesis and Protein PurificationA detailed description of the mutagenesis of sperm whale myoglobin was given by Carver et al.(1992). Briefly, the PstI-KpnI fragment of pMb413a (Springer and Sligar, 1987) was subcloned into pEMBL19 plasmids (Boehringer Mannheim) for mutagenesis, sequencing, and expression. The product of these manipulations was designated pEMbS-1. The Kunkel method of oligonucleotide-directed mutagenesis was used to construct the original L29F mutant. The double H64Q/L29F sperm whale mutant was then constructed using the E-helix cassette described by Springer et al.(1989) to make the original H64Q single mutant. The resulting plasmid was sequenced by the dideoxy method as described by Hattori and Sakaki(1986) using a United States Biochemical Corp. sequencing kit. Plasmids containing the mutant gene were transformed into the Escherichia coli TB-1 (Life Technologies, Inc.), expressed constitutively, and purified as described by Springer and Sligar(1987) (see also Lai et al.(1995)). The recombinant myoglobins were concentrated to 1 to 2 mM and stored in liquid nitrogen.Overall Kinetic MeasurementsAssociation and dissociation rates for O2 and CO binding were determined using conventional laser flash photolysis and stopped-flow rapid mixing techniques as described in detail by Rohlfs et al.(1990). Equilibrium association constants for O2 and CO binding were computed as the ratios of the overall rate constants. Autooxidation rates were measured using the techniques described by Brantley et al.(1993). Wild-type sperm whale myoglobin expressed in E. coli has been shown to be identical with native sperm whale myoglobin in terms of tertiary structure and kinetic properties (Springer et al., 1994). Thus, the numerical values presented for wild-type sperm whale myoglobin also apply to the native protein.Infrared and Resonance Raman SpectroscopyFor the infrared absorption measurements, approximately 30 μl of the MbCO solution (3-5 mM heme) was added slowly to a 1-mm cuvette to obtain a uniform film. The cuvette consists of 2 CaF2 windows separated by a 56-μm spacer and was purged with nitrogen gas immediately before the sample was added. Spectra were recorded at 2 cm−1 resolution in the region 1800-2100 cm−1 using a Mattson Galaxy 6020 spectrometer interfaced with a Compaq 386 computer. Up to 10,000 interferograms were collected for all samples and the corresponding buffer controls. The final IR spectra were corrected for buffer background by digital subtraction of the sample and control data.For the resonance Raman measurements, the reduced CO forms of the proteins were prepared by flushing deoxymyoglobin samples with 1 atm of CO under rigorously anaerobic conditions. The cyano-metmyoglobin samples were prepared by oxidation with ferricyanide and addition of potassium cyanide, and then excess KCN and K3Fe(CN)6 were removed by ion exchange chromatography. Approximately 100 μM samples were placed in a quartz spinning cell to prevent photodissociation in the case of MbCO and photoreduction in the case of Mb+CN (this was judged negligible as measured by the absence of any discernible contribution from the ν4 mode of the photoproduct). The resonance Raman spectra were acquired using a triple spectrograph (Spex 1877) equipped with a liquid nitrogen cooled 1152 × 298 pixel charge-coupled device (Princeton Instruments LN/CCD with an EEV chip) as the detector. Excitation was provided by the 415.4 nm output of a krypton ion laser (Coherent Innova 200-K3). The laser power was typically 5 milliwatts, and the spectral resolution was ∼4 cm−1.1H NMR MeasurementsCyano-metMb (cyano-metmyoglobin) samples for 1H NMR were made by adding K3Fe(CN)6 to oxidize the protein, followed by ion exchange chromatography to remove the ions and concentration in an Amicon ultrafiltration cell. The final solution was 4 mM heme in 50 mM NaCl, 10 mM KCN, 50 mM sodium phosphate, pH 8.6; 10%2H2O was added for the lock.All the 1H NMR spectra were collected on the GE Omega 500 MHz spectrometer. To effectively observe rapidly relaxing signals, the slowly relaxing diamagnetic envelope was suppressed by the WEFT pulse sequence (Gupta, 1976). Nonselective T1s for the resolved strongly relaxed protons were measured via the inversion-recovery experiment. Steady-state NOEs were recorded as described in detail previously (Emerson and La Mar, 1990a); the ratio of the steady-state NOEs to a common proton upon saturating the 1-CH3 and 8-CH3 groups was obtained from the ratio of the amplitudes of the NOEs in the difference traces (Rajarathnam et al., 1993). The phase-sensitive TOCSY (Braunschweiler and Ernst, 1983; Davis and Bax, 1985; Rance 1987), NOESY (Jeener et al., 1979), and magnitude COSY (Bax, 1982) measurements employed the same parameters, and were processed in the same fashion, as described in detail previously (Rajarathnam et al., 1992, 1993, 1994; Qin and La Mar, 1992).Magnetic Axes DeterminationThe magnetic axes were determined as described in detail previously (Emerson and La Mar, 1990b; Rajarathnam et al., 1992, 1993; Qin et al., 1993a). Experimental dipolar shifts for the structurally conserved proximal side of the heme were used as input to search for the Euler rotation angles, R(α, β, γ), that transform the molecular pseudosymmetry coordinates (x‘, y‘, z‘ or r, θ‘, Ω‘ (Fig. 1)) readily obtained from crystal coordinates into magnetic axes, x, y, z, by minimizing the global error function: F/n=Σ|δdip(obs)-δdip(calc)F(α,β,γ)|2(Eq. 1) where δdip(calc)=-13N[Δχax(3cos2θ-1)r-3+32Δχrhsinθcos2Ωr-3](Eq. 2) and δdip(obs)=δobs-δdia(Eq. 3) Δχax, Δχrh are axial and rhombic anisotropies, and δobs is the observed chemical shift referenced to 2,2-dimethyl-2-silapentane-5-sulfonic acid. δdia is the shift in the isostructural diamagnetic MbCO complex (Dalvit and Wright, 1987; Chiu, 1992) or calculated for protons whose δdia are not available by using the equation (Qin et al., 1993): δdia=δsec+δrc(Eq. 4) where δsec is the shift of an amino acid proton typical for α-helices, β-strand, coils, etc. (Wishart et al., 1991), and δrc is the heme-induced ring current shift of the proton based on the WT coordinates by using the eight-loop model (Cross and Wright, 1985). Minimizing the error function F/n in was performed over three parameters, α, β, γ, using available Δχax and Δχrh, or extended to all five parameters to yield both the Euler angles and anisotropies as described in detail previously (Rajarathnam et al., 1992).Distal Pocket Structure by NMRThe position of the E-helix was assessed by the relative intensities of the steady-state NOEs from the heme 1-CH3 and 8-CH3 to Val68(E11) CαH and Ala71(E14) CβH3, as described in detail previously (Rajarathnam et al., 1993). The orientation of Gln64(E7) was obtained by determining the sequential bond angles, starting with α-β, that optimally reproduced via and the R(α, β, γ), Δχax, Δχrh, δdip(obs) () for the residue, as described elsewhere in detail (Qin et al., 1993a). For Phe29(B10), the side chain orientation was determined separately by finding the sequential bond angles that correctly reproduce both the relative iron-induced relaxation (T1) and δdip(obs), as described previously (Qin et al., 1993a; Rajarathnam et al., 1993). Distances to the iron for proton a, RFe(a), was obtained from: T1(a)T1(b)=RFe 6(a)RFe 6(b)(Eq. 5) where a known proton, i.e. His(F8) NδH with RFe(a) = 5.0 Å and T1(a) = 31 ± 3 ms, yields RFe(b) when T1(b) is measured. The molecular modeling was carried out on a Silicon Graphics personal IRIS for the MbCO structure using the MIDAS program.X-ray CrystallographyCrystallization was carried out in concentrated ammonium sulfate solution using the batch method described by Phillips et al.(1990). The CO form of the double mutant crystallized in the hexagonal P6 space group with one molecule per asymmetric unit. Diffraction data were collected with a Rigaku R-AXIS IIC imaging plate system as described elsewhere (Quillin et al., 1993). The unit cell dimensions were a = b = 91.39 Å, c = 45.87 Å. The data set was 97% complete to a resolution of 1.7 Å, comprising 151,315 total measurements with 24,699 unique reflections. Rmerge was 10.9%.The coordinates of wild-type MbCO were used as the starting model in the refinement of the double mutant structure (Protein Data Bank entry 1MGK, Brookhaven National Laboratory; Quillin et al. (1993)). Initial difference maps were calculated using the program X-PLOR (Brunger et al., 1989). The Gln64(E7) and Phe29(B10) side chains were then introduced into the difference map using the molecular modeling program CHAIN (Sack, 1988). Constrained least-square refinements and map calculations were performed by X-PLOR using the Engh and Huber parameter set for the ferrous myoglobins (Engh and Huber, 1991). After several cycles of refinement, manual refitting, and solvent placement, the crystallographic R-factor converged to 17.3% with root mean square bond deviations of 0.02 Å. Coordinates for the L29F/H64Q MbCO structure have been deposited in the Brookhaven Protein Data Bank (1MCY).RESULTSO2and CO Binding and AutooxidationThe rate and equilibrium constants for ligand binding to wild-type, H64Q, L29F, and L29F/H64Q sperm whale and elephant myoglobins are listed in Table 1. he last column in this table lists the corresponding autooxidation rate constants. The H64Q substitution causes a marked increase in the rate of O2 dissociation due to weakening of hydrogen bonding between residue 64 (E7) and the bound ligand (Springer et al., 1994; Quillin et al., 1993). The net result is a 5-fold decrease in KO2IR and Resonance Raman SpectraInfrared spectra of CO bound to recombinant sperm whale and native elephant myoglobins are shown in Fig. 2. Wild-type sperm whale myoglobin shows two components in the IR spectrum of its CO complex, a major peak at 1944 cm−1 (A1,2 substrate) and a smaller one at 1932 cm−1 (A3 substrate, for a review see Li et al.(1994)). The L29F mutation causes a shift to a single component at νC-O = 1932 cm−1. The peak position for the H64Q mutant is the same as that of wild-type sperm whale myoglobin, but there is a loss of absorbance in the 1930 cm−1 region and an increase above 1950 cm−1. Both L29F/H64Q sperm whale and native elephant myoglobin show intermediate, more symmetrical peaks centered at 1938 cm−1.Figure 2:Fourier transform infrared spectra of CO-myoglobin complexes at pH 7, 25°C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Resonance Raman spectra of the CO-myoglobin and cyano-metmyoglobin complexes in the regions of the Fe-ligand stretching vibrations (νFe-CO and νFe-CN) are shown in Fig. 3and 4, respectively (for a review of these modes in hemoglobins and myoglobins see Yu and Kerr(1988)). The assignments for the νFe-CO and νFe-CN modes were confirmed via experiments with 13CO and 13CN- (viz. Ling et al. (1994)). The single L29F mutation causes an increase in νFe-CO from 509 for wild-type MbCO to 525 cm−1, and the single H64Q replacement causes a small decrease to 507 cm−1. When both substitutions are present, as in the double mutant and elephant myoglobin, νFe-CO is 513 cm−1. Similar results are observed for the Fe-CN complexes (Fig. 4). The L29F mutation causes a 7 cm−1 increase in νFe-CN; the H64Q replacement causes a 7 cm−1 decrease; whereas the νFe-CN values for the double mutant and elephant myoglobin are roughly equal to that of wild-type sperm whale myoglobin. Taken together, the spectral results in Fig. 2-4 argue strongly that elephant myoglobin contains a Phe29(B10) residue which interacts with bound ligands in a manner identical with that observed in the L29F/H64Q double mutant of sperm whale myoglobin. These data also emphasize the sensitivity of the Fe-CO and Fe-CN bond orders to amino acid substitutions in the distal pocket.Figure 3:Soret-excitation resonance Raman spectra of CO-myoglobin complexes at pH 7, 25°C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Soret-excitation resonance Raman spectra of cyano-metmyoglobin complexes at pH 7, 25°C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NMR Structural Studies: Assignment of Conserved ResiduesThe 500-MHz 1H NMR spectra for H64Q, L29F, 3The freshly prepared sample of L29F-cyano-metmyoglobin, initially prepared by adding CN-metMb, exhibited a single set of peaks indicative of a unique molecular structure. However, within a short time (1 day), a second set of peaks arise which finally account for ∼30% of the protein. These two sets of peaks likely arise from heme orientational disorder which is a characteristic equilibrium property of L29F-cyano-metmyoglobin, but not metMb. The presence of the second set of peaks leads to severe spatial overlap which, with the limited sample size available, restrict the extension of the two-dimensional NMR assignments to only those necessary to define the magnetic axes. and L29F/H64Q cyano-metmyoglobin in 2H2O are shown in Fig. 5. The nonlabile proton signals for these three proteins reflect a pattern similar to, but not identical with, that of wild-type sperm whale myoglobin and other point mutants (Rajarathnam et al., 1992, 1993; Qin et al., 1993a, 1993b). The significantly altered hyperfine shift pattern, as demonstrated earlier for other point mutants, must reflect changes in the magnetic axes of the ferric ion. The strategy for assigning the heme and conserved residues has been presented and discussed in detail previously for both sperm whale and Aplysia limacina cyano-metmyoglobin (Emerson and La Mar, 1990a; Qin and La Mar, 1992; Rajarathnam et al., 1992, 1993; Qin et al., 1993a, 1993b). Representative two-dimensional NMR data for L29F/H64Q cyano-metmyoglobin are shown to assign and characterize structurally the two mutated residues Gln64(E7) and Phe29(B10).Figure 5:Hyperfine-shifted portions of the 500-MHz 1H NMR reference spectra. A, H64Q-cyano-metmyoglobin at 35°C at pH 8.6 in 2H2O; B, L29F-cyano-metmyoglobin at 35°C at pH 8.6 in 2H2O; C, L29/H64Q-cyano-metmyoglobin at 35°C at pH 8.6 in 2H2O; D, in 1H2O; E, fast inversion recovery spectrum of L29F/H64Q-cyano-metmyoglobin in 1H2O collected at 12 s−1 to emphasize broad, efficiently relaxed proton signals. F, WEFT-NOE difference spectrum of L29F/H64Q-cyano-metmyoglobin upon irradiating 1-CH3 in 1H2O at 20°C. G, super WEFT-NOE spectrum of L29F/H64Q-cyano-metmyoglobin in 2H2O at 35°C at a repetition rate of 29 s−1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The 1H NMR spectrum of L29F/H64Q-cyano-metmyoglobin in 1H2O is shown in Fig. 5D. Two hyperfine shifted and strongly relaxed labile proton resonances are found in the low field window by comparison to the trace in 2H2O shown in Fig. 5C. One of these signals is apparent only in a partially relaxed spectrum in 1H2O which reveals a strongly relaxed NH proton overlapping the 1-CH3 signal that is absent in the same spectra in 2H2O (Fig. 5E). The super-WEFT trace of L29F/H64Q-cyano-metmyoglobin in 2H2O shown in Fig. 5G suppresses the diamagnetic envelope, retains the resolved strongly relaxed proton signals for CζH of Phe29(B10), CζH, C∊Hs of Phe43(CD1) and CγH of Ile99(FG5), and reveals strongly relaxed protons under the aromatic window, i.e. CδH of His97(FG3) and the ring CHs of His93(F8).Complete sets of resonances for the heme groups in all three mutants were assigned as described by Qin and La Mar(1992). Two series of sequential backbone NOESY cross-peaks uniquely locate the segments Leu89-Thr95(F4-FG1) and Lys63-Ala74(E6-E17) and the hyperfine shifted side chain signals (except for Gln64) are located by TOCSY and COSY (Qin and La Mar, 1992; Qin et al., 1993a, 1993b). Ile99(FG5) is identified by COSY and its NOESY contacts to the 4-vinyl and 5-CH3. His97(FG3) ring protons are identified by the characteristic short T1 (∼20 ms) for CδH and the dipolar contact of C∊H to 6Hα. A strongly relaxed (T1∼ 20 ms for CζH) AMM‘XX‘ spin system with NOESY cross-peaks to 5-CH3 locates the hyperfine shifted Phe43(CD1). TOCSY and MCOSY locate three weakly hyperfine shifted Phe side chains which characteristic NOESY cross-peaks to Phe43(CD1), Gln64(E7) CαH, and 2-vinyl/1-CH3 identify Phe33(B14), Phe46(CD4), and Phe138(H15), respectively (Emerson and La Mar 1990a; Rajarathnam et al., 1993). NOESY cross-peaks for 2-Hα and 3-CH3 to an upfield methyl peak at −0.44 ppm with apparent COSY cross-peaks to 1.0 and 0.6 ppm locate the C3H2-CδHs of Ile107(G8). Parallel studies on the two single chemical shifts for the heme model of L29F/H64Q cyano-metmyoglobin, as well as more limited data on the two single mutants, are listed in Table 2. 4The 9-proton input set I (references to set E in Rajarathnam et al.(1992)) includes CαH of Leu89(F4); CδH, C∊H of His97(FG3); CαH, CβH, Cα1H, Cγ2H, CγH3, CδH3 of Ile99(FG5). The 14-proton input data set II (referenced to as set D‘ in Rajarathnam et al.(1992)) has, in addition to the 9 protons identified above, CαH, CβH3 of Ala(F5) and CζH, C∊Hs, CδHs of Phe138(H15). The 18-proton input data set III (Qin et al., 1993a) has, in addition to the signals of data set II, CαHs of Gln91(F6), Ser92(F7), Ala94(F9) and CβH3 of Ala94(F9). For L29F-cyano-metmyoglobin, CαH of Ile99(FG5) and C∊H of His97(FG3) have not been assigned; instead of these protons, the C∊Hs, and CδHs of Tyr103(G4) have been used. Tabled 1 Open table in a new tab The proximal residues, Leu89(F4)-Ala94(F9), His97(FG3), Ile99(FG5), and His138(H15), of the double mutant exhibit heme-residue and inter-residue NOESY cross-peak patterns and paramagnetic induced relaxativities that are unchanged from those observed for wild-type cyano-metmyoglobin (Emerson and La Mar, 1990a; Rajarathnam et al., 1992). Thus, the structure on the proximal side of the heme group is highly conserved in all three mutants. For the most part, the unchanged distal residues also display intra- and inter-residue NOESY cross-peak patterns and paramagnetic relaxativity very similar to those observed for wild-type sperm whale myoglobin. The exceptions represent small structural changes required to accommodate the mutated E7 and B10 residues.Assignment of Gln64(E7) and Phe29(B10) ResonancesGln64(E7) in L29F/H64Q-cyano-metmyoglobin is detected in COSY spectra (Fig. 6A) as two spin fragments, NH-CαH CβH and CβH‘-CγH2, which are connected by a steady-state NOE between CγH and CβH (the CβH-Cβ‘H COSY cross-peak is too close to the diagonal to detect). The strongly relaxed (T1 = 13 ± 4 ms) labile proton detected under 1-CH3 in Fig. 5E yields NOEs to Gln64(E7) CαHs and Thr67(E10) CδH3 (Fig. 5F) These data place N∊H of Gln64(E7) at a distance of 4.4 ± 0.2 Å away from the iron (). The geminal partner N∊H‘ is too close to the water signal to detect. The complete Gln64(E7) assignment in the single H64Q mutant was obtained in a similar fashion.Figure 6:NMR spectra of of L29F/H64Q-cyano-metmyoglobin. A, the portions of the MCOSY spectrum in 1H2O, at 35°C showing spin connectivities for the Phe29(B10) and Gln64(E7). The MCOSY data are are processed by applying an unshifted sine-bell-squared window over 512 T1× 512 T2 points prior to zero-filling to 2048 × 2048 data points and Fourier transformation. B, the portions of the NOESY spectrum (τmix = 50 m" @default.
• W2114656295 created "2016-06-24" @default.
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• W2114656295 date "1995-09-01" @default.
• W2114656295 modified "2023-10-06" @default.
• W2114656295 title "A Double Mutant of Sperm Whale Myoglobin Mimics the Structure and Function of Elephant Myoglobin" @default.
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