(Received for publication, July 10, 1995)
From the
Recombinant tetrameric Hb
expressed and assembled in Escherichia coli has been
characterized extensively. Electrospray mass spectrometry and optical
and electron paramagnetic resonance spectroscopy suggest that the
overexpressed protein is identical to native human Hb. Although the
functional properties of this recombinant Hb are nearly identical to
native Hb, crucial differences exist between the two molecules. The
recombinant Hb expressed in E. coli has a lower Hill
coefficient even though oxygen equilibrium binding studies indicate
cooperative binding. The most significant difference observed between
the recombinant and native Hb is the loss of oxygen affinity regulation
by 2,3-diphosphoglyerate and protons. CO binding to the deoxy tetramer
was found to be biphasic with both phases sensitive to the presence of
allosteric effectors. The recombinant chains were isolated, and the
ligand binding properties demonstrated that the recombinant chains
behave in a similar fashion to native
and
. To investigate whether the chains were
capable of forming a well behaved tetramer, the isolated chains were
reassembled into a tetramer and purified to homogeneity. Oxygen binding
properties of the reassembled recombinant Hb now show an increased Hill
coefficient of 2.5, close to, but still slightly lower than, that
observed for native Hb. Additionally, reassembly of recombinant Hb
produces a protein that is subject to regulation by allosteric
effectors. Furthermore, CO binding to the reassembled recombinant deoxy
tetramer was found to be monophasic under all conditions.
Hb has long been studied as a model compound for many biochemical phenomena and continues to be the object of intense work to elucidate the molecular details of protein-protein recognition, allosteric regulation, ligand binding and dynamics, spectroscopy, energetics of cooperativity, and structure-function relationships(1, 2, 3) . X-ray structures of liganded (R), unliganded (T) states, and with several intermediate species have been solved to high resolution and have provided much information on the detailed characterization of the mechanism of action by Hb(4, 5, 6, 7) . In addition to mechanistic and structure-function studies, Hb has grown in popularity for use in clinical applications as a potential blood substitute(8) . These efforts, along with the possibility of engineering specific and novel properties into the molecule, have prompted the development of many recombinant Hb expression systems. We have investigated a purified recombinant human Hb using a coexpression system in Escherichia coli(9) in order to determine whether a completely assembled recombinant tetrameric Hb constitutively expressed in E. coli is fully functional.
The first
expression of individual human Hb chains in E. coli was
reported by Nagai and Thøgerson(10) . Their expression
system involved producing -globin as an insoluble fusion protein,
which was solubilized, purified, and cleaved with factor X
to produce the correct N terminus. The
-globin was then
reconstituted and refolded in the presence of native
chain to
produce a well behaved cooperative semirecombinant
tetramer(11) . Further characterization of this protein
including x-ray crystallography(12) , Raman spectroscopy, and
CO combination kinetics showed that the behavior of this
semirecombinant Hb was identical to that of native Hb(13) .
Individual recombinant
-globin has also been expressed in a
similar fashion and refolded with native
-globin to produce a well
behaved semirecombinant tetrameric Hb(14) . Recently, a
modified fusion protein system under chemical induction was used to
produce a recombinant
-globin which was then reconstituted into a
tetramer using a simplified method to produce a functional,
semirecombinant tetrameric Hb(15) . Our previous report has
shown that the
chain can also be expressed from cDNA clones as
insoluble aggregates using a T7 promoter without the use of a fusion
protein, thus circumventing some of the laborious problems associated
with a fusion protein expression system(9) . Unlike yeast (16, 17) and the fusion protein expression systems,
this construction leaves the initiator methionine on the N terminus.
Characterization of three N-terminal
chain mutants produced in E. coli using the T7 expression system (
1 + Met,
Val
Ala, and
Val
Met)
has been reported(18) . The addition of the initiator
methionine at the N-terminal of
chain produced a good
semirecombinant tetramer with only minor perturbations, but the removal
of the valine at the second position (
des Val) produced a
semirecombinant tetramer that was almost identical in allosteric
behavior, thermodynamic linkage, dimer-tetramer equilibrium, and CO
combination kinetics. In addition, x-ray structures of these mutants
indicated that the
des Val mutation had the least amount of
structural perturbation (19) and would serve as a good starting
point for further mutagenic studies.
Expression of a complete
recombinant soluble heme containing tetrameric Hb has recently been
achieved by coexpressing both and
chains in E.
coli(9, 20) . Initial characterization of a
soluble cytoplasmic heme-containing tetramer by coexpression of
and
chains from one plasmid in E. coli has been
performed(20) . The protein produced in these studies
co-migrated on an SDS-polyacrylamide gel electrophoresis with native Hb
(HbA) and had similar absorption spectra and chromatographic behavior
as HbA. However, oxygen equilibrium binding studies on this recombinant
Hb showed a reduced Hill coefficient, Bohr effect, and IHP (
)effect compared to native Hb, presumably due to the
presence of the initiator methionine.
To circumvent the inability of E. coli to processs the initiator methionine of both
recombinant Hb chains, we have constructed N-terminal mutations for
and
chains. The mutations remove the second amino acid,
valine, by removing the second codon triplet in the synthetic genes to
produce a recombinant Hb with
Val
Met and
Val
Met (Hb des Val). These modifications to
the N terminus should have minimal effect on a completely recombinant
Hb since the allosteric role of these residues is primarily associated
with the amino group rather than the side
chain(25, 26) . We have purified recombinant human Hb
coexpressed in E. coli to homogeneity and examined the
physical properties, using optical spectroscopy and EPR, and compared
them to native Hb. Functional properties investigated include oxygen
equilibrium binding to examine the oxygen affinity, cooperativity, pH
and allosteric effects of organic phosphates on oxygen binding. Carbon
monoxide binding to deoxygenated recombinant Hb was performed to
examine the T-state properties and allosteric effects of organic
phosphate and pH on CO binding kinetics. The integrity of the isolated
chains were examined using flash photolysis and displacement reactions
to determine their ligand binding properties. A completely recombinant
tetrameric Hb was reassembled from these isolated chains, purified and
its functional properties examined by measuring the kinetics of CO
binding to deoxygenated Hb, allosteric effects of organic phosphate on
the CO binding kinetics, and oxygen equilibrium binding in the presence
and absence of allosteric effectors IHP, DPG, and protons. The CO
combination kinetics provide an excellent means to probe the properties
of the deoxy or T-state Hb and its response to allosteric regulation
while the oxygen binding data provide a measure of cooperativity and
response of the reassembled Hb to allosteric regulation by organic
phosphate and pH.
A Kinetic Instruments (Bethesda, MD) stopped flow equipped with a 2-cm path cell and a 2-ms dead time was used. The temperature was controlled at 20 ± 0.1 °C with a Haake F3 circulating water bath and monitored with a thermistor. The absorbance changes at 420 and 435 nm were measured and stored as raw voltages on a Nicolet 3091 storage oscilloscope. The conversion of voltage to absorbance was performed using software written in our laboratory. The kinetic traces for native Hb were fitted with a single exponential to determine the observed rate. Recombinant Hb was fitted with either a single exponential or the sum of two exponentials.
Figure 1: Electrospray mass spectra of purified recombinant Hb (A) and native Hb (B).
Optical spectroscopy of recombinant human Hb
was used to probe the electronic environment of the porphyrin under
different ligation conditions. The deoxy spectra has a Soret maximum at
432 nm and a broad visible band at 556 nm. The oxy has a Soret maximum
at 415 and and
bands at 540 and 576 nm. Carbon monoxide
bound recombinant Hb has a Soret at 419 nm and
and
bands at
540 and 570 nm, respectively. The optical spectra of purified
recombinant Hb is identical to native Hb and corresponds well with
reported values(21) .
EPR spectra of both native and recombinant Hb were recorded at 7 K in 50 mM Tris-HCl, pH 8.9. The g values of 5.8 and 2.0 correspond to the high spin aqua bound complex. The g values at 2.5, 2.17, and 1.8 are for the low spin hydroxy complex. These data suggest that the recombinant Hb has ligand binding states identical to that of native Hb.
Oxygen equilibrium
binding curves of recombinant and native Hb were measured by the
continuous method described under ``Experimental
Procedures.'' Oxygen isotherms were measured in 50 mM Tris-HCl [Cl] = 0.1 M at 25 ± 0.1 °C. Fig. 2represents a typical
oxygen isotherm for native and recombinant Hb. Native Hb shows the
characteristic sigmoidal binding isotherm with a P
= 5.3 mm Hg and a Hill coefficient of 2.8. The binding
isotherm of recombinant Hb shows similar affinity relative to native
Hb; however, there appears to be a reduction in the Hill coefficient to
2.0 as manifested by a loss of the sigmoidal shape. Table 1lists
the oxygen equilibrium parameters for both the recombinant and native
Hb with and without organic phosphate and as a function of pH. The
interaction of the recombinant Hb with IHP produces a shift in the
P
identical to that found for native Hb but the
recombinant Hb still shows reduced cooperativity. The effect of DPG on
the recombinant Hb was most surprising. Native Hb oxygen affinity in
the presence of DPG shifts the P
from 5.3 to 12.9 mm Hg;
however, the recombinant Hb does not show a decrease in the oxygen
affinity in the presence of DPG, suggesting that DPG is incapable of
stabilizing the recombinant tetramer. In addition to organic phosphate
effects, pH effects on recombinant Hb equilibrium binding curves shows
only a 20% Bohr effect at pH 7.4. At pH 8.5, the recombinant Hb
displays a lower affinity than native Hb, while at pH 6.5 the
recombinant Hb exhibits a higher affinity compared to native Hb, and in
no case does the recombinant Hb show as high a degree of cooperativity
as native Hb.
Figure 2:
Oxygen equilibrium isotherm of native and
recombinant Hb. Conditions: 50 nM Tris-HCl, pH 7.4,
[Cl] = 0.1 M at 25 °C.
Protein concentration was 60 µM
heme.
Carbon monoxide binding kinetics were measured by stopped flow spectrometry by rapidly mixing CO with deoxygenated Hb. This technique starts with Hb in the deoxy state where virtually all the Hb is T-state tetramers and therefore the properties measured are that of the tetramer. The combination of CO with deoxygenated native and recombinant Hb was measured in the absence and presence of IHP. Fig. 3represents a normalized CO combination time course for native and recombinant Hb. The traces were fitted to a single exponential from which the observed rate was determined. CO combination to native Hb shows typical autocatalytic behavior, while CO combination to deoxy recombinant Hb shows two distinct kinetic phases. The time course for CO combination to recombinant Hb fits well to the sum of two exponentials where the fast phase made up as much as 40% of the total amplitude. The fast reacting component had a rate constant approximately one order of magnitude larger than that observed for native Hb, while the slow phase had a rate constant only two times larger than observed for native Hb.
Figure 3:
Normalized time course of CO combination
to deoxy recombinant Hb () and deoxy native Hb (
) in the
absence (A) and presence (B) of 2 mM IHP.
Observations were made using a 2-cm path length and monitoring at 420
nm. Solution conditions were 50 nM Bis-Tris, pH 7.0,
[Cl
] = 0.1 M at 20 °C.
Hb concentration was 2.5 µM heme after
mixing.
The IHP effect on the time course of CO combination to the recombinant Hb is also shown in Fig. 3, and the pH dependence with and without IHP is listed in Table 2. Even in the presence of saturating amounts of IHP, the time course of CO binding to the deoxy recombinant Hb remains biphasic, even when the pH is reduced to pH 6.0. The fast component shows an 85% reduction in the rate constant in the presence of IHP as the pH is lowered from 8.0 to 6.0. The slow component also displays a response to the presence of IHP and upon lowering the pH to 6.0. Unlike the fast component, the slow phase displays only a 50% reduction in the rate constant upon decreasing the pH from 8 to 6 in the presence of IHP. In the absence of organic phosphates, the recombinant Hb still remains biphasic with a monotonic decrease in the rate constant as the pH is lowered. The fast phase remains between 10 and 15 times faster than native Hb with rate constants similar to liganded Hb. The slow phase is similar to native Hb at pH 8.0; however, both the rate constants of the recombinant Hb are not as sensitive to the drop in pH as in native Hb.
Figure 4:
Flash photolysis of (A) and
(B) chains. Normalized time course for CO recombination
to recombinant (
) and native (
) chains. The reactions were
carried out in 0.1 M phosphate, pH 7.0, at 20 °C and
monitored at 420 nm. The protein concentration was 5 µM and CO concentration was 50
µM.
where k is the bimolecular second order
association rate constant and k
is the
dissociation rate constant for CO. The resultant pseudo first order
rate constants determined from the fit depend linearly upon the amount
of carbon monoxide present. The CO on rate, k
, of
the recombinant
and native
chains were found to be
identical within experimental error (Table 3). Similarly the CO
association rate, k
, of recombinant
chain
is also linearly dependent upon the ligand concentration and the
calculated rate constants are identical to that of native
-globin.
The dissociation rate constants for both native and recombinant
and
chains were determined by ligand displacement reaction
with NO. In these experiments the protein-CO complex was mixed with a
solution of NO which exhibits a higher affinity and a smaller
dissociation rate constant than does CO. The concentration of NO, the
replacing ligand, is high, and the observed rate is described by:
where k` and k
are
the association and dissociation rate constant for the carbon monoxide
and k`
is the association rate constant of NO.
For dissociation of CO from the
and
complexes, 1 atm of NO
gas was mixed with the protein. Since k`
k`
for all heme proteins (29) at
high concentrations of NO the observed replacement rate constant
determined is directly equal to the dissociation rate of CO
((k`
[CO]/k`
[NO] 1). Under the conditions used, the observed rate
approximates the off rate of CO. The rates determined are listed in Table 3. The off rate determined for comparison of the chains
indicate that there is little difference between either the recombinant
or
chains and the native
or
chains. Equilibrium
constants determined by k
/k
confirm that the
recombinant chains are similar to native chains.
Figure 5:
Oxygen isotherm of native and reassembled
recombinant Hb (A) with conditions described in Fig. 2.
CO combination kinetics (B) of deoxy reassembled recombinant
HB () and deoxy native Hb (
) with conditions as described in Fig. 3A.
Since the reassembled recombinant tetrameric Hb appears to show oxygen binding and cooperativity similar to native Hb, the CO combination kinetics to the reassembled deoxy tetramer were investigated to determine if the high affinity R-state-like hemes had been reorganized into a more well behaved tetramer. Fig. 5represents typical time course of CO combination to the reassembled recombinant tetramer and to native Hb. The curves of the reassembled tetramer were nearly monophasic and fit well to a single exponential. The traces show a substantial loss in high affinity hemes that was observed for recombinant Hb but still did not show true autocatalytic behavior. Table 5lists the calculated rate constants as a function of pH and organic phosphate. The rate constant of the recombinant reassembled Hb are quite similar to native Hb. The response to organic phosphate and protons suggest that this material is very similar to native Hb.
While the physical
characterizations of recombinant Hb are similar to native Hb, the
O binding properties are markedly different. Oxygen
equilibrium binding properties of recombinant Hb provide an overall
means to assess oxygen affinity, cooperativity and response to pH and
organic phosphate. The Hill coefficient determined in this study is
lower than other values reported for a similar assembled recombinant
tetrameric Hb produced in E. coli(20) but the oxygen
affinities are similar. Reduction of the Hill coefficient indicates
destabilization of the tetramer possibly by either alteration of the
dimer-tetramer equilibrium or perturbation within the
interface.
Stabilization of the
tetramer and regulation of oxygen affinity by organic phosphates and
protons at secondary sites is critical for Hb function, and altered
allostery for recombinant Hb may be indicative of small but significant
structural changes. Since organic phosphates such as IHP and DPG both
bind at the 1 N terminus (30, 31) and are
critical for allosteric function, these effectors may be sensitive to
perturbations due to the valine to methionine substitutions or
alterations which perturb the binding site. Since the substitution of
Val
Met does not affect the allosteric
regulation by organic phosphate, the observed alterations in organic
phosphate binding of the completely recombinant Hb could imply changes
within the central cavity of the recombinant Hb. Interaction with IHP
suggests that these perturbations or rearrangements do not appear to
sterically hinder the binding of organic phosphate but may affect the
electrostatic interactions. The alterations in electrostatic
interaction may possibly explain the reduction in DPG stabilization,
while IHP stabilization is apparently unaffected. In addition
significant reduction in the Bohr effect of the recombinant Hb strongly
suggests that structural rearrangements may have occurred during in
vivo assembly resulting in a change in the environment of the Bohr
groups.
CO combination kinetics of deoxy Hb suggest that the deoxy recombinant Hb exists as a heterogenous population of at least two different reactive hemes. The slow phase of the recombinant Hb has a rate constant similar to native Hb suggesting one population may be a reasonably well behaved deoxy or T-state tetramer. The fast kinetic phase of the recombinant Hb may be a heterogeneous population of species which are unable to form a well behaved deoxy tetramer. The second order rate constant for the fast component is approximately a factor of 10 greater than that of a native Hb deoxy tetramer, but within a factor of 2-3 for combination of CO to R-state Hb (32) or to isolated chains(21, 33) .
Allosteric effectors were applied in the kinetic analysis to ascertain whether the biphasic behavior of the recombinant Hb was kinetically sensitive to IHP and pH. CO combination to deoxy recombinant Hb shows no change in the shape of the traces or the return to autocatalytic behavior upon addition of IHP or a drop in pH. The fast phase is sensitive to allosteric effects suggesting that this population is in a tetrameric form since dimers and isolated chains are not sensitive to IHP effects(34) . The kinetic data of the recombinant Hb suggest that the deoxy tetramer exists in at least two distinct conformations one with normal deoxy kinetics and the second which is R-state like or high affinity. These data are also consistent with the equilibrium binding data which indicated alterations which destabilize the tetrameric recombinant Hb.
The molecular origin of
these perturbations may lie within the assembly of the tetramer
presumably within the interface
and/or disruption of the deoxy salt bridges of the deoxy conformer. Hb
Bethesda (
Tyr
His), a mutant which alters the
normal salt bridges around the C terminus and destabilizes the deoxy
structure has a high oxygen affinity, small response to DPG and IHP,
and a significant reduction in cooperativity. Olson and Gibson (35) have studied the kinetic properties of this variant. CO
combination to deoxy Hb Bethesda exhibits biphasic kinetics with a fast
component similar to that of isolated chains. They demonstrated that
the observed fast kinetics of Hb Bethesda is due in part to an
alteration in the deoxy structure to a more liganded like Hb. Hb
Chesapeake (
Arg
Leu) a variant within the
interface, also shows biphasic CO
combination kinetic behavior and this variant may exist in two deoxy
conformations not in rapid equilibrium(36) . By comparison with
natural occurring variants, origin of perturbations associated with the
recombinant Hb may lie within the in vivo assembly of the
tetramer during expression in E. coli. Since interactions at
the contacts between the dimers mediate cooperativity, and structural
perturbations at the
interface may
perturb cooperative interactions, incorrect formation of either the
dimer or assembly of the dimers into the correct quaternary structure
is an interpertation consistent with the data. Assembly of the
recombinant Hb which has high affinity heme may be a result either of
misfolding of the tertiary structure of the subunits which results in
formation of an altered quaternary structure, or assembly of correctly
folded subunits into a stable alternative high affinity quaternary
structure.
These results indicate that
there is a great similarity between both the recombinant and native
and
chains, although some small differences exist which
tends to rule out the hypothesis that the recombinant des Val
and
chains are misfolded. These small changes are not indicative of
drastic changes within the heme pocket or the porphyrin environment.
These data are consistent with the spectroscopic data of the
recombinant Hb which indicate no discernible difference in the ligation
state of the heme iron. The recombinant chains do not appear to inhibit
the bound iron ligand complex, but rather appear to adequately
accommodate the ligand in the heme pocket in a similar fashion as
native Hb.
An entirely different picture emerges when one
compares the oxygen binding properties of the reassembled tetramer versus unmodified recombinant Hb in the presence of
heterotropic allosteric effectors. IHP effects of the reassembled
recombinant Hb are similar to native HbA as would be expected because
of the high affinity of IHP for the deoxy tetramer. In contrast the
specific effect of DPG on the reassembled tetramer indicates a profound
change to a lower oxygen affinity identical to that of native Hb. That
the reassembled recombinant tetrameric Hb is affected by DPG may imply
a rearrangement of the amino acid side chains within the central cavity
that results in tighter binding of DPG and therefore an increase in
stabilization of the tetramer. The increase in the Bohr effect of the
reassembled tetramer to a more native-like behavior again implies a
possible rearrangement of the reassembled recombinant Hb. The return of
regulation by DPG and increase in the Bohr effect of the reassembled
tetramer again argues against the substitution of Val Met as the
cause of the reduced heterotropic effect observed for the recombinant
Hb, but rather provides support that the large side chain at this
position is relatively innocuous to the heterotropic regulation of Hb
and suggests a misassembly of the tetramer expressed in E.
coli.
The kinetics of CO combination to deoxy Hb probe both the quaternary structure and the heme binding site of Hb. The results of the experiments from the reassembled recombinant Hb show a monophasic time course and fit well to a single exponential. This monophasic behavior did not change under varying conditions. Response of the reassembled tetramer to allosteric regulation by organic phosphates and protons provides an additional means to kinetically probe the functional behavior of the reassembled tetramer. The reassembled tetramer has a 60% reduction in the rate of CO binding upon addition of IHP while the native has a 55% reduction in the rate. In addition the reassembled tetramer has a reduction in the rate with lowering of pH similar to that of native Hb. The appearance of monophasic behavior and the loss of the fast reacting component after reassembly of the tetramer further suggests that a rearrangement of the quaternary structure of the tetramer has occurred during the reassembly to produce a more native-like protein capable of carrying out full functional behavior.
Expression of recombinant Hb in E. coli produces
a protein with a reduced cooperativity, loss of most response to
allosteric regulation and mulitphasic kinetics. It is clear that
reassembly of the tetramer significantly reduces the heterogeneity
associated with the recombinant Hb produced in E. coli and
tends to restore correct functional behavior including allosteric
regulation. The apparent correct functional behavior of the chains
suggests that the placement of the subunits in the quaternary structure
of the recombinant Hb expressed in E. coli is altered thereby
not having full functional behavior. By isolating the subunits, and
reassembling the tetramer we have shown that a functional tetrameric Hb
is produced. In vivo expression of Hb requires that both
and
chains be present (9) suggesting that template
assisted folding between the two chains is required for proper folding
and assembly of the tetramer. During expression, misassembly could
occur due to misalignment of the template. This misassembly, however,
does not produce an unstable Hb but rather produces a soluble
tetrameric Hb physically similar to but functionally distinct from that
of native Hb. This is quite unique among oligomeric proteins which
misassemble, since these proteins usually precipitate into inclusion
bodies. It has been known that some oligomeric proteins lack the
inherent ability to correctly assemble into a protein with biological
function. In many cases proteins are expressed as insoluble aggregates
in inclusion bodies, solublized in urea, then renatured and reassembled
to form an active protein. Molecular chaperons are required, in some
instances, to assemble these proteins into the correct structure. For
example, plant ferrodoxin-NADP
oxidoreductase has
recently been expressed in the presence of GroEL and GroES to produce
an assembled native peptide, which when expressed in a
chaperon-deficient strain of E. coli the protein failed to
assemble properly(39) . The large and small subunits of
ribulose-bisphosphate carboxylase/oxygenase have also been coexpressed
in a soluble form in E. coli, but the specific activity is
lower than expected, presumably due to the inability to form a
completely assembled complex(40) . Bacterial luciferase, an
heterodimer, has been produced by expressing the individual
peptides separately in E. coli, and assembling the dimer after
each of the individual subunits is purified(41) . The
heterodimer that formed by this procedure failed to assemble into an
active heterodimer, suggesting that the subunits had folded into a
catalytically incompetent structure. Activity of bacterial luciferase
was restored by incubating the heterodimer in the presence of 5 M urea(42) .
The recombinant Hb expressed in E. coli may exist with multiple quaternary structures or conformations which are not in equilibrium with each other, and the perturbations are most notable in the deoxy or T-state structure. The structures are presumably similar to the native structure but not completely identical and may exist in a local energy minimum. By isolating the subunits and reassembling the tetramer the quaternary structures are interconverted to a more native-like form. This study, however, was unable to distinguish between a misassembled quaternary structure, or misfolded subunits which lead to misassembly, or both. Thus reassembling the recombinant tetramer provides a mechanism to produce site-directed mutants for detailed structure-function studies using a coexpression system for recombinant Hb.