From the Department of Chemistry, University of California, Davis, California 95616
From the Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892
From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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The bivalve mollusc Lucina pectinata
harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a
monomeric hemoglobin I, HbI, with normal O2, but
extraordinarily high sulfide affinity. The crystal structure of aquomet
Lucina HbI has revealed an active site with three residues
not commonly found in vertebrate globins: Phe(B10), Gln(E7), and
Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M.,
Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86-89). Engineering these three residues into sperm whale myoglobin
results in a triple mutant with ~700-fold higher sulfide affinity
than for wild-type. The single crystal x-ray structure of the aquomet
derivative of the myoglobin triple mutant and the solution
1H NMR active site structures of the cyanomet derivatives
of both the myoglobin mutant and Lucina HbI have been
determined to examine further the structural origin of their unusually
high sulfide affinities. The major differences in the distal pocket is
that in the aquomet form the carbonyl of Gln64(E7) serves
as a H-bond acceptor, whereas in the cyanomet form the amido group acts
as H-bond donor to the bound ligand. Phe68(E11) is rotated
~90° about 2 and located ~1-2 Å closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially
eliminates the stabilizing interaction with sulfide and, together with
the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of
Lucina HbI.
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INTRODUCTION |
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Despite substantial amino acid sequence variability, the
reversible binding of molecular oxygen in myoglobin
(Mb)1 and hemoglobin (Hb) is
achieved by a surprisingly invariant protein folding topology: a heme
group imbedded within 7-8 packed -helices (1, 2). Examination of
the more than 300 known sequences of vertebrate Hbs and Mbs and the
>130 nonvertebrate globin-like sequences demonstrates that only two
residues appear to be conserved, the proximal His at helical position
F-8 and the Phe parallel to the heme surface at an interhelical loop,
position CD1 (1, 3). The residue considered to be pivotal in the
stabilization of the bound O2 is the one whose side chain
can provide a neutral donor for a hydrogen bond with the ligand, such
as His or Gln at the distal position E-7. Among vertebrate Mbs and Hbs,
His predominates, with Gln occurring only in elephant Mb (4), shark Mb
(5), hagfish Hb (6), and the
chain of opossum Hb (7). Although His
is also found in the majority of nonvertebrate Hbs and Mbs, Gln is not
uncommon and, in addition, other residues may occupy the E-7 position,
e.g. Val in Aplysia (8) and several other mollusc
Mbs (9), Tyr in the Mb from Paramphistomun epiclitum (10,
11), and Leu in the monomeric components of Glycera Hb (12).
Although capable of O2 binding, a number of the variant globins exhibit some anomalous functional properties. Parallel functional and structural studies on genetically engineered mutants of
mammalian Mb have shown that changes in only a very limited number of
residues in the distal pocket often transfers the essence of the
unusual behavior of the natural genetic variant to the appropriate
reference protein mutant (13-17).
Cytoplasmic Hbs occur in many symbiotic associations between bacteria and invertebrates or plants and may have a physiological role in the symbiosis (18-20). The bivalve mollusc Lucina pectinata, found in sulfide-rich coastal sediments, harbors sulfide-oxidizing chemoautotrophic bacteria (21, 22). Its abundant cytoplasmic hemoglobins consist of three single-chain components, each having a moderately high affinity for oxygen (P50 = 0.1-0.2 torr), which is achieved, however, by a very different balance of combination and dissociation rates. Reactions of the monomeric HbI with oxygen are rapid, whereas those of HbII and HbIII are extraordinarily slow (23).
Recent crystal structures of Lucina aquometHbI and the sulfide bound form (24, 25) have revealed a distal Gln64(E7) and Phe29(B10), neither of which is rare among invertebrate Mbs, Hbs. However, the E-11 position is uniquely occupied by Phe rather than the aliphatic amino acids Val, Ile, or Leu. To examine the structural origin of strong sulfide binding in Lucina HbI, we have systematically replaced His64(E7), Leu29(B10), and Val64(E11) with Gln, Phe, and Phe, respectively, in sperm whale myoglobin and then determined the structures of the resulting multiple mutants by NMR and x-ray crystallography. This approach is similar to that used to study the unusual functional properties of Aplysia Mb, Ascaris Hb, and elephant Mb, and provides comparison between the native protein and the synthetic mimic in two ligation/spin states and between protein structures in crystal and solution (13-17, 26, 27). The NMR studies were pursued to define more carefully the positions of the labile protons of Gln64(E7) and their role as hydrogen bond donors to the bound ligand. Both the paramagnetic relaxation and induced dipolar shifts in metMbCN and metHbICN can be used to model the position of distal residues (14-17, 26, 29, 30). The combined NMR and crystallographic results reveal systematic differences between the orientation of Gln64(E7) in the aquomet and cyanomet complexes of Lucina HbI and L29F/H64Q/V68F-Mb, which demonstrate that Gln64(E7) may serve as either a H-bond acceptor or donor to bound ligands and that a difference in the orientation of the Phe68(E11) ring may account for the difference in sulfide affinity between Lucina HbI and the sperm whale triple mutant Mb mimic.
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EXPERIMENTAL PROCEDURES |
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Protein Preparation-- The pUC19 plasmid containing V68F sperm whale myoglobin was made by Egeberg et al. (31). The plasmid containing L29F/H64Q/V68F sperm whale Mb was constructed from pEMBL19 containing L29F Mb using cassette mutagenesis as described in Springer et al. (32). Vectors harboring the mutated gene were transformed into Escherichia coli strain TB1 and expressed constitutively using a 100-liter fermentor. The resulting soluble holomyoglobin protein was purified as described previously (33, 34).
Protein Crystallization-- Crystals of the recombinant L29F/H64Q/V68F-Mb were grown by the batch method (35, 36) in a temperature-controlled environment (17 °C). Large single crystals developed out of solutions ranging from 2.4 to 2.7 M ammonium sulfate buffered at pH 9 (20 mM Tris-HCl, 1 mM EDTA).
X-ray Data Collection and Structure Determination--
Crystals
were hexagonal, space group P6, with one molecule per asymmetric unit
as originally solved by Phillips et al. (35). They were
mounted in sealed quartz capillary tubes prior to data collection. A
single data set was collected from one crystal at room temperature
using a Rigaku R-axis IIc imaging plate system and copper K
radiation from a Siemens rotating anode operated at 50 mV and 90 mA.
The unit cell dimensions of the L29F/H64Q/V68F-metMbH2O were a = 91.39, c = 45.75. A total of
74,100 measured reflections from 60 images (
= 1.5°) with an
Rmerge of 5.7% for all data were reduced to
15,526 unique intensities (5.0 to 1.85 Å, 87.3% complete) using the
program XDS.
Crystal Structure Refinement--
Starting coordinates were
generated using the L29F/H64Q-MbCO (Protein Data Bank entry 1MCY,
Brookhaven National Laboratory) (17) to calculate initial phases. The
coordinates for the additional mutated residue (V68F) were built from
an electron density omit map. Solvent atoms were introduced in positive
density peaks over four . Cycles of conventional positional
refinement were carried out with XPLOR (37-39) alternated with manual
fitting using the CHAIN software package (40). The crystal structures
were all refined with Engh and Huber (41) topology and parameters with no restraints on the iron atom to remove bias in metal position. The
crystallographic refinements converged to an R-factor of
16.8% for the triple mutant metmyoglobin structure with root mean
square bond deviations of 0.017 Å. Coordinates have been deposited in the Protein Data Bank (entry 1OBM).
1H NMR Measurements--
All the 1H NMR
spectra were collected on the GE Omega 500 MHz spectrometer. The
strongly relaxed signals were optimally detected in water-eliminated
Fourier transform spectra (42). Nonselective T1s for the
resolved strongly relaxed protons were measured via inversion-recovery
experiment. Steady state NOEs were recorded as described previously
(43). The phase-sensitive TOCSY (44, 45), NOESY (46), and conventional
magnitude COSY (MCOSY) (47) employed the method described by States
et al. (48) to provide quadrature detection in the
t1 dimension. Solvent suppression, when
required, was achieved by direct saturation in the relaxation delay
period. 512 blocks were collected with 25.0 kHz spectral widths to
include all resonances, and 10 kHz to improve resolution for the
diamagnetic envelope. 128 to 256 scans were accumulated with repetition
rate of 0.7 s1 or 1.2 s
1 for each block
with free induction decays of 2048 complex points. The data were
processed as described previously (49); details are given in the figure
captions. All two-dimensional data were processed on Silicon Graphics
workstation using the software package Felix from Biosym/MSI (San
Diego).
Magnetic Axes Determination--
The magnetic axes were
determined as described previously (15, 16, 29, 30, 50). Experimental
dipolar shifts for the structurally conserved proximal side of the heme
were used as input to search for the Euler rotation angles,
(
,
,
), that transforms the molecular pseudo-symmetry
coordinates (x', y', z', or
R,
',
' (Fig. 1))
readily obtained from crystal coordinates (17, 24, 25, 51) into
magnetic axes, x, y, and z, by minimizing the
global error function,
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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Dipolar Shift Simulations-- The position of a substituted or perturbed residue can be determined by minimizing a local error function. This local error function, designated F*(residue)/n' to distinguish it from that global error function in Equation 1, is given by Ref. 30,
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(Eq. 4) |
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(Eq. 5) |
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RESULTS |
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Hydrogen Sulfide and O2 Binding to Lucina HbI and Sperm Whale Mb
A brief summary of the affinities of the myoglobin mutants and Lucina HbI for O2 and hydrogen sulfide2 is shown in Table I (17, 23, 28). Lucina HbI shows a 5,000-fold higher affinity for sulfide than recombinant wild-type sperm whale myoglobin, whereas the two proteins have similar affinities for oxygen. There are progressive increases in KH2S as the active site of the whale myoglobin is altered to resemble that of the mollusc hemoglobin. All three single mutations, L29F, H64Q, and V68F produce large, 6-20-fold increases in sulfide affinity, and the effects on KH2S are roughly additive in the double and triple mutants. In contrast, these mutations produce markedly different effects on O2 affinity. For example, the single H64Q and L29F mutations produces a 5-fold decrease and 15-fold increase, respectively, in O2 affinity. As a result, KO2 for the triple mutant is only 3-4-fold higher than wild-type myoglobin and very similar to that of Lucina HbI (Table I).
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High Resolution Crystal Structure of Sperm Whale L29F/H64Q/V68F-metMbH2O
The three mutated side chains in the recombinant myoglobin do not alter the overall tertiary structure of myoglobin nor do they appear to sterically hinder the bound ligand. Two of these mutated residues, Gln64(E7) and Phe68(E11), occupy positions equivalent to those observed in the structures of corresponding H64Q-Mb and V68F-Mb single mutants (36, 55). In contrast, the aromatic ring at position 29 in the triple mutant is perpendicular to the orientation found in sperm whale L29F- and L29F/H64Q-MbCO (Fig. 2) (17, 56). A detailed comparison of the distal pockets of the L29F/H64Q/V68F-metMbH2O and Lucina metHbIH2O (24) is presented in Table II and Fig. 2. The overall positions of the three mutated residues in the recombinant sperm whale metmyoglobin are close to those of the corresponding residues in the clam protein, but the exact orientations of the phenyl side chains are significantly different. The distal glutamines in both proteins form hydrogen bonds with the coordinated water molecule.
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Heme Pocket Structure of L29F/H64Q/V68F-metMbCN and Lucina metHbICN
Resonance Assignments-- The 500 MHz 1H NMR spectra for L29F/H64Q/V28/F-metMbCN, V68F-metMbCN, and Lucina-metHbICN in 1H2O at 25 °C are shown in Fig. 3, A-C, respectively. The signals for the heme for each protein were located and assigned by the characteristic pattern of dipolar contacts about the heme periphery among the TOCSY identified vinyl, propionate groups, and the pyrrole methyl, meso-Hs in a fashion standard for both diamagnetic and paramagnetic heme proteins. The pattern of heme chemical shifts is similar to that observed in wild-type sperm whale metMbCN (not shown). Resonances for residues near the heme are assigned to the degree possible, as limited by spectral congestion and paramagnetic relaxation, and by standard backbone dipolar connectivities together with characteristic TOCSY and/or COSY connectivities for several side chains (15, 16, 49). Other residues not addressable by these approaches are assigned on the basis of dipolar contacts to the heme and/or to other assigned residues. Inasmuch as this assignment strategy has been reported in detail for both wild-type and mutant metMbCN (17, 29, 30, 43), two-dimensional data are shown only as relevant to the placement of perturbed distal pocket residues in both protein complexes.
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Magnetic Axes Determination--
All dipolar shifted residues
except Phe68(E11) exhibit a good correlation between
dip(obs) and the slope of the chemical shift in a Curie
(chemical shift versus T
1) plot that is
indicative of well defined orientations to the heme (not shown) (49). A
variety of proximal proton dipolar shifts which correlate with their
Curie slopes for each protein were
used.3 To determine the
magnetic axes, both as three-parameter searches for
(
,
,
)
using the wild-type metMbCN anisotropies,
ax = 2.04 × 10
9 m3/mol,
rh =
0.48 × 10
9 m3/mol (29), and as
five-parameter searches for both
(
,
,
) and the anisotropies.
Both mutants exhibit excellent correlation between
dip(obs) and
dip(calc) with very low
residual F/n in Equation 1 (not shown) typical of
previous NMR studies of mutant metMbCNs (15, 16, 29, 30, 50). The
chemical shifts for numerous distal residues are also well predicted.
The resulting Euler angles using different input data sets were highly
clustered for each protein, although distinct for each of the proteins. For L29F/H64Q/V68F-metMbCN, the optimized angles (and ranges for the
various fits) are: a tilt of the major axes from the heme normal,
~ 6.3° (6.1-6.6°), direction of tilt,
~
40° (
40° to
50°) and rhombic axes,
~
+
= 30° (20° to
40°) using MbCO crystal coordinates. The magnetic axes for
V68F-metMbCN are
= 5° (0-10°),
= 7.5° (7-8°), and
= 40° (30-50°). The optimized anisotropies for each five-parameter
fit differ inconsequentally for those of wild-type metMbCN and yield
Euler angles within the ranges obtained by the 3-parameter fit (not
shown). When the coordinates for L29F/H64Q/V68F-metMbCN are taken from
the L29F/H64Q/V68F-metMbH2O crystal structure presented
herein, we obtain values for the angles and anisotropies that are well
within the ranges obtained using the wild-type coordinates. The
magnetic axes (and ranges in their values), obtained similarly for
Lucina metMbICN, using either the Lucina
metHbIH2O (24) or metHbIH2S (25) crystal
coordinates, are
= 7° (6-8°),
= 155 (150-160),
~
+
= 255° (250-260°), and optimized anisotropies are
unchanged from those of wild-type metMbCN (not shown).
Orientation of Distal Residues in
L29F/H64Q/V68F-metMbCN--
Using either the wild-type MbCO crystal
coordinates with the mutated Phe29(B10) inserted with an
orientation as found in the crystal structure of L29F/H64Q-MbCO
(half-open squares in Fig.
6A), or directly the crystal
coordinates of the presently characterized
L29F/H64Q/V68F-metHbH2O (open squares in Fig.
6A), a qualitative, but not quantitative, correlation
between dip(calc) and
dip(obs) are
observed, and the RFe (C
H = 4.7 Å) is shorter than indicated by T1 = 33 ms
(RFe = 5.1 ± 0.3 Å). However, altering
1 by 5° from that in the L29F/H64Q-metMbCO and a 50°
rotation of
2 leads to a Phe29(B10)
orientation whose
dip(calc) correlate very well with
dip(obs) (closed squares in Fig.
6A) and for which the RFe
(C
H) = 5.1 Å is consistent with the
T1 value.
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Orientation of Distal Residues in Lucina MetHbICN--
The
orientations for Gln64(E7) in the Lucina
metMbH2O (24) or metHbIH2S (25) crystal
structures yielded completely unacceptable fits for
dip(obs) versus
dip(calc) for
Lucina-metMbICN in solution (as shown by open
circles and half-closed circles, respectively, in Fig.
6B); the correlation is particularly poor for the labile N
Hs where both models predict even the wrong sign for
dip(calc). Moreover, the low-field N
H
closest to the iron has RFe ~3.5 Å and 5.2 Å in the two crystal structures while the observed
T1 = 12 ms predicts RFe ~ 4.2 ± 0.2 Å via Equation 5. Since the Gln64(E7)
orientation in Lucina metHbIH2O had the
N
H oriented toward the ligated water, which is expected
to be a H-bond donor, the Gln orientation with
3 rotated
by 180° to interchange the carbonyl and amide groups was considered.
This altered orientation leads to different
N
H2 dipolar shifts (not shown) that fit
dip(obs) better, but still unacceptably. Attempts to
obtain a fit for
dip(obs) by sequential
1,
2,
3 rotation failed
since
dip(calc) for C
Hs is quite
insensitive to the
1. Instead, the combination of the
large low-field
dip(obs) and RFe ~ 4.2 Å (from the T1 = 12 ms) was used to
uniquely locate the low-field N
H in the crystal
coordinates, and a search made for the position in space of the other
N
H (with intra N
H distance 1.88 Å) to
satisfy both the upfield
dip(obs) and
RFe ~ 4.6 ± 0.2 Å obtained from its
T1 = 22 ms. Upon obtaining a reasonable fit for
the N
Hs that satisfy both relaxation and
dip(obs) constraints, a search was
pursued for the range of
1,
2, and
3 allowed by the fixed C
H and
NH2 positions. The good correlation between
dip(calc) and
dip(obs) for this optimized
Gln orientation is shown in Fig. 6B (closed
circles). The resulting tortional angles for Gln64(E7)
are listed in Table II where they are compared with values in the
crystal structure of Lucina metHbIH2O.
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DISCUSSION |
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Solution NMR Cavity Structure-- The conventional two-dimensional NMR approach allowed unambiguous assignment for all relevant residues in the heme cavity. The intrinsic paramagnetism of cyano-metMb, instead of being an impediment to assignment, was actually an advantage since the dipolar shift with minimal relaxation provide significantly enhanced resolution for the E and F helical segments. For the proximal side of the heme, the NOESY cross-peak patterns and paramagnetic relaxation for the two Mb mutants were indistinguishable from those observed for wild-type metMbCN and predicted by the wild-type MbCO structure. A similarly strongly conserved proximal structure is confirmed for Lucina metHbICN. These conserved proximal structures allow the determination of meaningful magnetic axes for each protein. The robust nature of the magnetic axes for L29F/H64Q/V68F-metMbCN is confirmed by the indistinguishable magnetic axes determined using either the wild-type MbCO or triple mutant crystal structures.
For the distal residues, whose relaxation properties, dipolar shifts and/or NOESY cross-peak pattern differed from that expected for the reference crystal structure, the combined restraints for dipolar shifts and paramagnetic relaxation allow the determination of the their orientations in a more definitive manner than allowed by the NOESY cross-peak pattern themselves. The accuracy of the distal orientations is limited only by the basic assumption that the helical backbone positions are inconsequentially perturbed in the mutant and/or ligation variant relative to the available crystal structure. It is noted that the labile protons of the distal Gln64(E7) are readily assigned. The combined effect of the dipolar shift and paramagnetic relaxation allow the placement of the labile protons in the distal pocket so as to more accurately describe hydrogen bonding between Gln64(E7) and the bound ligand than is possible in some of the crystal structures. The aromatic ring for Phe68(E11) is oriented away from the expected binding site with the ring inserting into a largely pre-existing cavity in the distal pocket (55). The absence of a methyl group at theComparison of Distal Pockets in Solution and Crystal
Structures--
For all but select distal residues, the heme pocket
structure for each protein is essentially the same in cyanomet and
aquo-met derivatives. In the case of the sperm whale triple mutant, the major difference in the Gln64(E7) orientation between the
cyanomet and aquomet complexes is ~180° rotation about
3 that interchanges the carboxy and amide termini (Table
II, Fig. 2A). The 180° rotation of the Gln side chain
terminus is completely consistent with the stabilizing roles of the
side chain for the different ligands. For the cyano complex, the
N
H2 is oriented to serve as a H-bond donor
to bound cyanide, whereas in the aquomet complex, the carbonyl oxygen
is oriented to accept a H-bond from coordinated water. The small
changes in
1,
2 for Gln64(E7)
(Table II) help to accommodate the larger cyanide ligand.
Implications for Sulfide Binding-- All three single distal pocket substitutions in sperm whale Mb cause an enhancement of sulfide binding but have variable effects on oxygen affinity. The key mutation is H64Q, which causes a 25-fold increase in sulfide affinity by itself. The triple mutant, L29F/H64Q/V68F-metMb, has a sulfide affinity which is ~700 times greater than that of wild-type metMb and only ~7-fold less than that of Lucina HbI. Thus the triple mutant exhibits an increased sulfide binding free energy of ~3.8 kcal/mol or about ~75% of the stabilization of the bound sulfide (~5.1 kcal/mol) by native Lucina HbI relative to that by wild-type sperm whale Mb. Hence, a major portion, but not all, of the remarkable sulfide affinity of Lucina HbI can be transferred to a mammalian Mb by replacing the three residues in contact with bound sulfide with those found in Lucina HbI. The high affinity of Lucina HbI for sulfide was attributed (24, 25) to a combination of the hydrogen-bond acceptance by the Gln64(E7) side chain carbonyl, the hydrophobic pocket provided by the "cage" generated by the three Phe side chains, B10, CD1, and E11, and highly favorable electrostatic interactions between the sulfur and the edges of the three aromatic rings. While the distal pockets for the triple mutant Mb and Lucina HbI are similar (Fig. 2), there are some significant differences that relate to their differences in affinity for sulfide.
The side chains of Gln64(E7) and Phe43(CD1) have similar orientations in the sperm whale triple mutant and the clam protein (Fig. 2C). However, both Phe29(B10) and Phe68(E11) in the sperm whale mutant are rotated ~90° about ![]() |
ACKNOWLEDGEMENTS |
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We thank Eileen Singleton for the expression and purification of the triple mutant myoglobin, Mike Berry for help in the protein's crystallization and subsequent structural refinement, and Jun Qin for assistance with NMR experiments.
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FOOTNOTES |
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* This work was supported by United States National Institutes of Health Grants HL16087 (to G. N. L.), GM35649 and HL47020 (to J. S. O.), AR40252 (to G. N. P.), and Postdoctoral Fellowship AR08355 (to E. A. B.), the States of Texas Advanced Technology Program Grant 003604-025 (to G. N. P. and J. S. O.), Robert A. Welch Foundation Grants C-612 (to J. S. O.) and C-1142 (to G. N. P.), and the W. M. Keck Center for Computational Biology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed.
§ Current address: Somatogen, Inc., Boulder, CO 80301.
1
The abbreviations used are: Mb, myoglobin; Hb,
hemoglobin; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; MbCO,
carbonmonoxymyoglobin; MCOSY, magnitude COSY; metMbCN, cyanomet
myoglobin; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser
effect; NOESY, two-dimensional nuclear Overhauser spectroscopy; ppm,
parts per million; TOCSY, two-dimensional total correlation
spectroscopy; V68F-Mb, Val68(E10)Phe-Mb;
L29F/H64Q/V68F-Mb, Leu29(B10)
Phe,
His64(E7)
Gln, Val68(E11)
Phe-Mb; HbI,
monomeric hemoglobin component I; metHbICN, cyanomet-hemoglobin
I.
2 E. A. Brucker, G. N. Phillips, R. Lile, J. S. Olson, R. F. Eich, and J. B. Wittenberg, unpublished results.
3
The dip(obs) used for the
magnetic axes for L29F/H64Q/V68F-metMbCN and V68F-metMbCN are:
Leu89(F4) C
H; Ala90(F5)
C
H, C
H3;
Phe138(H15) C
Hs, C
Hs,
C
H; Ile99(FG5) C
H,
C
H, C
H, C
H',
C
H3, C
H3; His97(FG3) C
H, C
H; and those
used for Lucina metHbICN are: Ala62(E5)-Val72(E15),
Phe92(F4)-Ala98(F10), Arg101(FG1),
Gly102(FG2), Ile104(FG4),
Ala108(G7)-Phe110(G9), Val130(H11), and
Ala131(H12).
4 M. Bolognesi, personal communication.
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REFERENCES |
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