The Role of Water Molecules in the Association of
Cytochrome P450cam with Putidaredoxin
AN OSMOTIC PRESSURE STUDY*
Yoshiaki
Furukawa and
Isao
Morishima
From the Department of Molecular Engineering, Graduate School of
Engineering, Kyoto University, Kyoto 606-8501, Japan
Received for publication, November 9, 2000, and in revised form, January 12, 2001
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ABSTRACT |
We have investigated the osmotic pressure
dependence of the association between ferric cytochrome P450cam and
putidaredoxin (Pdx) to gain an insight into the role of water
molecules in the P450cam-reduced Pdx complexation amenable to
physiological electron transfer. The association constant was evaluated
from the electron transfer rates from reduced Pdx to P450cam. The
natural logarithm of the association constant Ka
was linearly reduced by the osmotic pressure, and osmotic stress yields
uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the
association in the nonphysiological redox partners P450cam and oxidized
Pdx. Although general protein-protein associations proceed through
dehydration around the complex interface, the interfacial waters could
mediate hydrogen-bonding interactions. Therefore, about 10 more
interfacial waters imply an additional water-mediated hydrogen-bonding
network in the P450cam·reduced Pdx complex, which does not exist in
the complex with oxidized Pdx. It is also possible that the
water-mediated hydrogen-bonding interactions support a high P450cam
affinity for reduced (Ka = 0.83 µM
1) relative to oxidized
(Ka = 0.058 µM
1) Pdx.
This study points to a novel role of solvents in assisting redox
state-dependent interaction between P450cam and Pdx.
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INTRODUCTION |
The function of cytochrome P450cam
(P450cam)1 is to catalyze the
stereoselective oxidation of camphor to 5-exo-hydroxycamphor (1). The full catalytic cycle of P450cam starts with NADH, which
reduces the FAD-containing protein putidaredoxin reductase (PdR). PdR
then transfers an electron to putidaredoxin (Pdx), and two distinct
electron transfer (ET) steps are needed from Pdx to P450cam to enable
catalysis. In the catalytic cycle, ferric P450cam tightly binds the
reduced form of Pdx (Pdxred), which is a catalytically
competent complex, with a dissociation constant Km
of 1.6 µM (2), whereas the product after intracomplex ET
(ferrous P450cam and oxidized Pdx (Pdxox)), easily
dissociates into the free forms (Km = 88 µM) (2). Although the redox state-dependent
affinity of P450cam with Pdx is necessary to efficiently inject an
electron into the P450cam active site, the molecular mechanism of the
recognition between P450cam and Pdx has not yet been proven in detail.
In addition to being an electron shuttle to P450cam, Pdx is a
conformational effector of P450cam (3). Low potential iron-sulfur
protein such as spinach ferredoxin and bovine adrenodoxin can transfer a first electron to P450cam but not a second electron, resulting in a
very slow turnover of the P450cam monooxygenase reaction. Therefore,
many investigators have tried to characterize the association between
P450cam and Pdx.
As yet, site-directed mutagenesis studies of amino acid residues on Pdx
and P450cam have indicated that salt bridges are formed between
Asp38 (Pdx) and Arg112 (P450cam) and
Asp34 (Pdx) and Arg109 (P450cam) upon
complexation (4-8). Computer modeling studies also support the
importance of salt-bridge and hydrogen-bonding interactions upon
P450cam-Pdx association (9, 10). Sligar and co-workers (2, 4, 11) have
reported the mutational studies on the C-terminal residue
Trp106 in Pdx and proposed that the presence of a
C-terminal aromatic residue is required for the complex formation
between P450cam and Pdx. Because a contribution to association energy
from an aromatic residue is generally expected to come from desolvation of the residue in the complex, the elimination of water molecules from
the P450cam·Pdx complex interface is plausible. Such a partial desolvation of the aromatic residue in the binding cleft could accompany the decrease in the dielectric constant for the binding region, which results in the increase of the magnitude of any existing
electrostatic interactions. Therefore, not only the simple salt-bridge
and hydrogen-bond formation, but also the water molecules at the
P450cam·Pdx complex interface should be important for molecular recognition between P450cam and Pdx. However, little attention has so
far been paid to the role of water molecules in the P450cam-Pdx association reaction.
Because bound water molecules appear to form an integral part of the
structure of proteins and their complexes with partner proteins,
solvent has been shown to play a significant role in biologically
important protein association systems. For example, upon
ferredoxin-ferredoxin:NADP+ reductase association, the
large positive entropy changes (+157 J mol
1
K
1) with small, unfavorable enthalpy changes were
observed by Jelesarov and Bosshard (12). Most protein-protein
associations in the ET reactions show large positive entropy changes of
binding by excluding water molecules from the complex interface
(13-15). In contrast, our group (16) has reported that the energetics
of the P450cam-Pdx association is characterized by a favorable enthalpy change (
H =
53.8 kJ mol
1) and a
negative entropy change (
S =
93.2 J
mol
1 K
1) of binding. With respect to the
negative entropy change in the P450cam-Pdx association, it is plausible
that the water molecules are trapped rather than excluded at the
complex interface between P450cam and Pdx.
To gain insights into the molecular recognition between P450cam and
Pdx, we examined the contribution of interfacial water molecules to
P450cam-Pdx association by using the osmotic stress strategy (17).
Decreasing the water activity of the bulk solution by using osmolytes
can shift the equilibrium between the unbound proteins and its complex
because protein-protein interfaces behave as though they are separated
from the bulk solution by a semipermeable membrane, the protein itself.
That is, high osmotic pressure promotes dehydration of the molecular
surface and should promote binding if water is released from the
complex interface. Similarly, if interfacial water stabilizes binding,
a decrease in water activity will decrease the binding affinity.
Di Primo et al. (18) have reported the osmotic pressure
dependence on the NADH consumption rate in the P450cam catalytic cycle
and observed the decreased turnover under high osmotic pressure. However, a number of steps in the P450cam catalytic cycle inhibit the
detailed characterization of the relationship between P450cam-Pdx association and the water molecules. In this study, we measured the
rate of the interprotein ET from Pdxred to ferric P450cam,
which is a more specific measurement of the association constant of the
physiological complex, ferric P450cam·Pdxred.
Furthermore, by spectrophotometric examination of the osmotic pressure
dependence of the nonphysiological association between ferric P450cam
and Pdxox, the role of water molecules in the recognition
of P450cam to Pdx is discussed.
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EXPERIMENTAL PROCEDURES |
Preparation of Cytochrome P450cam and Putidaredoxin--
P450cam
was expressed in Escherichia coli strain JM83 having the
pUC18 vector and purified with the procedures previously described
(19). Purified preparations with an RZ value
(A391/A280) greater than
1.4 were employed in this study. Pdx was expressed in E. coli strain RR1 as described elsewhere (8).
Measurement of the Electron Transfer Rate Constants--
Laser
flash photolysis experiments utilized the third harmonic (355 nm) of a
Q-switched Nd:YAG laser, which provides photolysis pulses with a
half-peak duration of 10 ns. The monitoring beam was generated by a
xenon lamp (150 watt) and focused on the sample cell at the right angle
of the excitation source through a notch filter. The transmitted light
was detected by a photomultiplier that is attached to a monochrometer,
UNISOKU USP-501. A two-channel oscilloscope (TDS320) was used to
digitize and accumulate the signals that were transferred to a NEC
PC-98 computer for the further analysis. Temperature was controlled by
using a circulating water bath.
Scheme 1 shows the photochemical reaction
in which the acridine triplet and the methyl viologen (MV) initiate
protein-protein ET (20). Briefly, a laser flash at 355 nm excites
acridine to its triplet state, which in turn oxidizes EDTA, resulting
in formation of acridine semiquinone in less than 1 µs. Following
that, the photoreduced acridine reduces methyl viologen (MV), to form
the MV cation radical. Then, the MV cation radical reduces
Pdxox some hundred times faster than ferric P450cam in the
mixture of P450cam and Pdx (See "Results"). Finally, the ET
reaction from Pdxred to ferric P450cam can be monitored as
the formation of ferrous carbonyl P450cam (P450cam-CO) under CO
atmosphere. Sample solutions for the laser experiments were degassed
and purged with CO under vacuum. Because the reaction product,
P450cam-CO, is accumulated by laser flash, we adopted the data of the
kinetic trace from one laser flash for further analysis. The
experiments were repeated for five times to estimate the experimental
error. The kinetic measurements were performed at 293 K in a
CO-saturated solution containing 10 µM Pdxox,
5-25 µM ferric P450cam, 15 µM acridine,
500 µM MV, 25 mM EDTA, 50 mM
potassium phosphate, 1 mM d-camphor, pH 6.7.
Preparation of Sample Solution Buffers--
Preparation of the
solution buffers containing cosolvents for laser experiments were
accomplished as follows. 50 mM potassium phosphate buffers
containing 12, 16% (w/w) glycerol, 8, 16% (w/w) ethylene
glycol, or 15, 30% (w/w) glucose, were prepared. Acridine and
d-camphor were completely dissolved in the aqueous buffer. The concentration of acridine in the solution buffers was checked spectrophotometrically (
353 = 17 mM
1 cm
1). Following that, MV
and EDTA were added, and the pH of the solution was checked. In the
case of the buffers containing glucose, the pH of the solution was
decreased to about 6.0 with the addition of EDTA. Because the pH of the
other cosolvent-containing solutions was 6.7, we adjusted the pH of
glucose-containing buffers to 6.7 with concentrated KOH solution.
Analysis of the Electron Transfer Rate Constants--
To obtain
the association and the ET rate constants between ferric P450cam and
Pdxred, we followed the analysis method of Davies and
Sligar (2). The three-state model appropriate to the Pdx-P450cam enzyme
system is shown in Reaction 1.
In this reaction scheme, Pdxred and ferric P450cam
are the associating species A and B. Species C corresponds to the
reactant complex, and D represents the bound and unbound product
proteins, Pdxox and P450cam-CO. As previous studies have
shown (21), the bimolecular association step is sufficiently faster
than the ET step in the complex in the reaction between P450cam and
Pdx. In such a case, the observed rate constants of the formation of
P450cam-CO can be expressed by the sum of the concentrations of each
unbound reactants at equilibrium as in the following equation,
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(Eq. 1)
|
where K
= (k1/k
1) is the
association constant between ferric P450cam and Pdxred. It
has been reported that the value of k
ET is
essentially zero in the experimental system used in this study because
the presence of CO effectively prevents the reversal of this reaction
(21). The summed equilibrium concentrations of Pdxred and
ferric P450cam were estimated from the amplitude of the reaction progressive curves. Briefly, the concentrations of Pdxred
generated by a laser pulse, [Pdxred]o, was
estimated in the absence of P450cam from the absorbance change at 420 nm (Fig. 1A), a wavelength at
which there is a significant bleach in the (Pdxred minus
Pdxox) redox difference spectrum (
420 =
5.83 mM
1 cm
1). The
P450cam-CO concentration, [P450cam-CO], generated by the ET
reaction with Pdxred was also determined by the absorbance
change at 446 nm (Fig. 2), where
P450cam-CO dominantly absorbs (
446 = 106.6 mM
1 cm
1). Because the ET
reaction between Pdxred and ferric P450cam occurs through
the 1:1 complex (9, 21, 22), the concentration of Pdxred or
ferric P450cam at equilibrium can be expressed as follows,
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(Eq. 2)
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(Eq. 3)
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where [ferric P450cam]o is the concentration of
ferric P450cam in the sample before a laser pulse.

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Fig. 1.
Transient absorbance changes at 420 nm
obtained after a 355-nm laser excitation of Pdxox
(A) and ferric P450cam (B) at 293 K. The sample solution contains 10 µM protein in 50 mM KPi, 1 mM d-camphor,
15 µM acridine, 500 µM MV, 25 mM EDTA, pH 6.7
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Fig. 2.
Transient absorbance change at 446 nm
obtained after a 355-nm laser excitation under CO-saturated atmosphere
of 15 µM ferric P450cam
in the presence of 10 µM
Pdxox. The residuals of the single-exponential
fitting are shown above the signals. The experiments were performed at
293 K, and the proteins were dissolved in 50 mM
KPi, 1 mM d-camphor, 15 µM acridine, 500 µM MV, 25 mM
EDTA, pH 6.7. Inset, CO partial pressure dependence of the
observed rate constants. The dashed line shows the CO
rebinding rate constants of P450cam.
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Measurement of the Dissociation Constant of Ferric P450cam with
Pdxox--
The methods of Hintz et al. (22)
were used to measure the dissociation constant of ferric P450cam with
Pdxox. The sample cuvette contained 1500 µl of 50 mM potassium phosphate, pH 6.7, containing 1 mM
d-camphor with cosolvents and 2 µM ferric P450cam. The reference cuvette also contained 1500 µl of the same solution except for P450cam. About 5 mM Pdxox
was added stepwise to both cuvettes up to 25 µl. The measurements were carried out at room temperature. The binding of Pdxox
to ferric P450cam induces a spectral change that is characterized by a
decrease and increase in absorption around 390 and 420 nm, respectively. This spectral change indicates a Pd-induced spin-state shift of the ferric P450cam from high-spin to low-spin. The free Pdx
concentration, [Pdx]free, was calculated by Equation 4 (23).
|
(Eq. 4)
|
The reciprocal of the absorbance change at 391 nm was then
plotted against the reciprocal of free Pdxox
concentrations, and the spectroscopic dissociation constant, Ks, was calculated.
Analysis of the Association Step in the Presence of
Osmolytes--
To examine the effects of the osmotic pressure on the
association constants Ka between P450cam and Pdx, we
used three kinds of osmolytes as performed by Di Primo et
al. (18); glycerol, ethylene glycol, and glucose. The osmotic
pressure, Posm, for the solvent at different
osmolyte contents was estimated using Equation 5,
|
(Eq. 5)
|
where XH2O and
VH2O are the mole fraction and the molar
volume of water, respectively. XH2O was calculated using
tabulated values of the water content for each osmolyte/water mixtures
(24). For VH2O, a value of 18 ml/mol was used. To quantitatively characterize the osmotic pressure
dependence of the association process between P450cam and Pdx, we
estimated the volume change associated with the osmotically available
water in the two states of the equilibrium,
VW, which is defined by Equation 6
(25).
|
(Eq. 6)
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 |
RESULTS |
Upon flash photolysis of acridine/EDTA/MV solution, we found an
absorbance increase at 700 nm in a µs time scale, which is the
wavelength maximum for the MV cation radical (data not shown). In the
absence of an electron-accepting species, P450cam or Pdx, the MV cation
radical was stable for more than 500 ms. The MV cation radical
can reduce Pdxox about 100× faster than ferric P450cam
(20). For example, 1.5 µM MV cation radical can reduce
Pdxox at 1250 s
1 in the presence of 10 µM Pdxox, whereas the reduction of 10 µM P450cam occurs with a much slower rate constant, 15 s
1 (Fig. 1). Concomitant with the reduction of
Pdxox or ferric P450cam, the MV cation radical was oxidized
to its original state, MV, with a similar rate constant to the
reduction rate of Pdxox or ferric P450cam, showing that the
MV cation radical donates an electron to Pdxox or
ferric P450cam (data not shown). The faster reduction of
Pdxox over P450cam by the MV cation radical was confirmed
in the all cosolvent concentrations examined in this study.
Even in the solution containing both Pdxox and P450cam, an
electron was favorably transferred from the MV cation radical to Pdxox. In the presence of 10 µM
Pdxox in the sample, MV cation radical decays with the same
rate constant, regardless of the absence or the presence of P450cam.
This decay rate is accelerated about 2-fold, when the solution contains
20 µM Pdxox. These data imply that the MV
cation radical first reacts with Pdxox, and the favorable
reduction of P450cam by MV cation radical is less possible under the
experimental conditions in this study.
In addition, we observed the redox process of Pdx to ensure the
favorable reduction of Pdx by MV cation radical in the presence of
P450cam. Lacking Pdx in the sample solution, only the increase of the
absorbance at 467.4 nm, which is the isosbestic point between ferrous
P450cam and P450cam-CO,2 was
observed by a laser shot, indicating the formation of P450cam-CO by
laser-generated MV cation radical. When the reaction mixture further
contains 10 µM Pdx, the absorbance changes at 467.4 nm initially decrease by laser shot and show the subsequent gradual increase (Fig. 3A). Because
there is significant bleach at 467.4 nm in the static difference
spectrum of reduced minus oxidized Pdx, the initial decrease by laser
shot indicates the reduction of Pdx. The rate constants of the
reduction of Pdx are essentially the same as those of the MV cation
radical decay estimated from the absorbance changes at 700 nm (Fig.
3B), which indicates that MV cation radical favorably
donates an electron to Pdxox even in the presence of the
excess P450cam (10 µM Pdx and 20 µM
P450cam). The gradual increase of the 467.4 nm absorption means both of
the re-oxidation of Pdxred and the formation of
P450cam-CO. The single exponential function was adequate to fit this
absorption increase, which implies that Pdxred donates an
electron to P450cam and the ET from MV cation radical to P450cam have
little contribution to the observed kinetics.

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Fig. 3.
Transient absorbance change at 467.4 nm
(A) and 700 nm (B) obtained after a
355-nm laser pulse. The residuals of the double-exponential
(A) or the single-exponential (B) fitting are
shown at the top of the figure. Conditions; 10 µM Pdxox, 20 µM P450cam in 50 mM KPi, 1 mM d-camphor,
15 µM acridine, 500 µM MV, 25 mM EDTA, pH 6.7.
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From these results, the MV cation radical primarily reduces
Pdxox in the mixture of P450cam and Pdx, as suggested
previously (20). This aspect of reduction reactions by MV cation
radical will reflect the difference in the electrostatic potentials on
the surface near the redox center of each protein (26). The best
position on the surface of P450cam for ET reactions is a positive
charged domain, whereas Pdx carries a negative charge near the redox
center [2Fe-2S] cluster. Because the MV cation radical bears the
positive charge, the favorable electrostatic interaction with
negatively charged Pdx makes the reduction faster because of a closer
spatial distance between redox centers.
The kinetic trace shown in Fig. 2 illustrates the formation of
P450cam-CO under CO-saturated conditions in the presence of 15 µM ferric P450cam and 10 µM
Pdxox. Many studies have clarified that the equilibration
of Pdxred·ferric P450cam complex is fast relative to the
ET process in the complex (Reaction 1, Refs. 21, 22). Also, the
CO-binding step in ferrous P450cam will not be rate-limiting under the
conditions of saturated CO used in this study (21). As shown in the
inset of Fig. 2, the observed rate constants are independent
of CO partial pressure in the range between 0.6 and 1.0 atm. Whereas
the CO-binding to P450cam becomes a limiting step in the low CO partial
pressure, this result clearly indicates that the experimental condition of the saturated CO atmosphere permits the observation of the ET
process from Pdxred to P450cam.
We also confirmed little effect by the small molecules (MV, acridine,
glycerol, ethylene glycol, and glucose) on the CO binding process in
P450cam under the saturated CO atmosphere. A laser pulse dissociates CO
from P450cam-CO, which is prepared by addition of dithionite, to form
ferrous P450cam. Following that, ferrous P450cam rebinds CO with a
rate constant of 70 s
1 in the 50 mM
potassium phosphate/1 mM d-camphor, pH 6.7. Even when the cosolvents are present, the CO rebinding rate constants remain
virtually unchanged; 70 s
1 in 500 µM MV/15
µM acridine, 16% ethylene glycol, or 30% glucose, 60 s
1 in 16% glycerol. The CO-binding process is,
therefore, not affected by the cosolvent concentrations used in this
study. The P450cam-CO formation rate shows saturation behavior
against the concentration of P450cam (Fig.
4), which is more evidence that the
CO-binding step is not rate-limiting. Therefore, the formation of
P450cam-CO in this experiment follows the events of the ET reaction
step in the Pdxred·ferric P450cam
complex.3

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Fig. 4.
The dependence of the observed rate constant
on the summed equilibrium concentration of Pdxred and
ferric P450cam in the free states. Circle, the absence
of the cosolvent; square, 12% (w/w) glycerol;
triangle, 16% (w/w) glycerol. The solid curve is
the least squares fit to the data using Eq. 1 (See "Experimental
Procedures").
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Association of Ferric P450cam with Pdxred--
The
kinetic traces of P450cam-CO formation can be well fitted by a
single-exponential function (Fig. 2), which is also the case for the
traces in all cosolvent concentrations. By plotting the observed rate
constants as a function of the sum of Pdxred and ferric
P450cam concentrations at equilibrium (Eq. 1, Fig. 4), we obtained an
association constant, K
, and
an ET rate constant, kET, in the ferric
P450cam·Pdred complex under various cosolvent
concentrations as summarized in Table I.
The obtained values of kET and
K
without cosolvent molecules
are consistent with those reported previously (2, 6). The addition of
cosolvents decreases the association constant,
K
, and has little effect on
kET. K
changed from 0.83 µM
1 (in the absence of
cosolvents) to 0.19 µM
1 (16% ethylene
glycol), whereas the changes in kET are small
and in the range between 37 and 45 s
1. The effects of
cosolvents are, therefore, mainly on the association process but not on
the ET process. Fig. 5A shows
the plot of association constant against osmotic pressure, and ln
K
scales down as a linear
function of osmotic pressure (R2 = 0.903), which
means that the increase in osmotic pressure destabilizes the complex
between Pdxred and ferric P450cam. However, the poor
correlations with the changes in
K
were found against the
other solvent colligative properties, such as the viscosity
(R2 = 0.235), the dielectric constant
(R2 = 0.507), or the water molarity
(R2 = 0.662). In addition, the variety of
cosolvents (glycerol, ethylene glycol, glucose) tested rules out a
specific interaction between the cosolvent molecules and the
P450cam·Pdx complex. Osmotic stress (Eq. 6) yields a value for
VW of 457 ± 99 cm3/mol.
Assuming a partial molar volume of water equal to 18 cm3/mol, the obtained value, 457 ± 99 cm3/mol, would correspond to 25 ± 6 molecules of
osmotically active water involved in the association process.
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Table I
Association constants, K and K
and electron transfer rate constants, kET, between ferric
P450cam and reduced Pdx under various cosolvent concentrations
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Fig. 5.
Dependence of the association constants on
the osmotic pressure for ferric P450cam binding to Pdxred
(A) and Pdxox (B) for the
three osmolytes: glycerol (circle), ethylene glycol
(square), glucose (diamond), and in
the absence of cosolvent (cross). The solid
line represents the best fits by linear regression using Eq. 6.
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Association of Ferric P450cam with Pdxox--
Fig.
6 shows the Pdxox-induced
difference spectra for ferric P450cam without cosolvents. Complexation
with Pdxox decreases the absorbance around 390 nm and
increases the absorbance around 420 nm. This Pdx effect has been
observed previously (27) and serves as an indicator of complex
formation between ferric P450cam and Pdxox. As depicted in
the inset to Fig. 6, in all cosolvents used, the reciprocal
of the absorbance decrease at 391 nm is linearly correlated with the
reciprocal of the concentration of free Pdxox. In the
absence of any cosolvents, the spectroscopic dissociation constant,
Ks, was 17.2 ± 1.4 µM, which
is consistent with the previously reported values (28, 29). By adding
the cosolvents, glycerol, ethylene glycol, and glucose,
Ks was increased, as summarized in Table I. As
seen in the case of ferric P450cam-Pdxred association,
linear correlation was also observed between the natural logarithm of
the association constant in ferric P450cam/Pdxox
(K
= 1/Ks) and the osmotic pressure
(R2 = 0.923). The ln
K
is poorly correlated with
the other solution properties; R2 = 0.464 (relative viscosity), 0.746 (dielectric constant), 0.846 (water
molarity). By using Eq. 6, we obtained 230 ± 30 cm3/mol as
VW, and the number of
trapped osmotically labile water molecules can be estimated as about
13 ± 2 upon association of ferric P450cam with
Pdxox.

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Fig. 6.
Difference spectra of 2 µM ferric P450cam with
addition of Pdxox at room temperature in 50 mM KPi, 1 mM
d-camphor, pH 6.7. The spin-state shift of ferric
P450cam from high- to low-spin induced by the binding of
Pdxox is clearly observed. The final concentration of
Pdxox is 86 µM. Inset,
double-reciprocal plot of titration of ferric P450cam with
Pdxox in the absence of cosolvents. Details are described
under "Experimental Procedures."
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 |
DISCUSSION |
As shown in Table I, addition of the cosolvents to the solution
leads to an increase in the osmotic pressure, which disfavors the
interactions between ferric P450cam and Pdx irrespective of the redox
state. Previous studies have revealed that the osmotic pressure
perturbs the spin equilibrium of ferric P450cam and favors the
high-spin form by excluding the water molecules in the P450cam active
site (30). The dehydrating effect of increasing osmotic pressure on
ferric P450cam (camphor-bound) was also observed in this study (data
not shown). Because the high and low spin forms of ferric P450cam
represent two conformational states of the protein (31), it is possible
that the P450cam conformational changes induced by osmotic pressure
reduce the association between ferric P450cam and
Pdxox/red. In such a case, the osmotic pressure dependence
of the association of ferric P450cam would be the same between ferric
P450cam·Pdxox and ·Pdxred complexes. The
different volume changes, however, are observed for ferric
P450cam·Pdxred (
VW = 457 cm3/mol) and ferric P450cam·Pdxox
(
VW = 230 cm3/mol), which implies
that the osmolyte-induced conformational changes are not the major
origins of the reduced association.
Moreover, the substantial conformational changes might be expected to
influence the ET rate constant in the ferric
P450cam·Pdxred complex, because the ET rate constant is
very sensitive to the distance and the orientation between the two
redox proteins. According to the Marcus theory (32), which is often
applied to the analysis of the protein ET reactions, only the 0.5 Å elongation of the distance is predicted to result in the 2-fold
reduction in the ET rate constant. Against such an expectation, the
observed ET rate constant is almost unchanged up to 10 MPa (Table I),
showing that the osmotic pressure makes a smaller perturbation on the structure of the P450cam·Pdx complex. As to Pdx, its UV/vis spectrum is not affected by osmotic pressure, and the redox potential is also
unchanged (data not shown). It is, therefore, unlikely that the reduced
association between ferric P450cam and Pdxox/red is caused
by the protein conformational changes induced by osmotic pressure.
Application of osmotic pressure relies upon the existence of a
water-permeable barrier (the complex interface in this study) separating a population of water molecules from a nonpermeating solute,
osmolyte (17). Correlation of a process with osmotic pressure implies a
change in the size of this population. Previous studies have shown that
most protein-protein association processes in the ET reactions are
usually accompanied by the release of water molecules from the
interface of the complex through the hydrophobic interactions (12,
33-36). The increase of osmotic pressure leads to the increase of its
association constants because of the desolvation at the complex
interface, as seen in the cytochrome b5-cytochrome c association reaction
(
VW =
47 cm3/mol) (34).
Therefore, the simplest interpretation of the reduced association by
osmotic pressure is that a population of bound waters, sequestered from
bulk solvent, is present at the P450cam·Pdx complex interface but is
not present in the free states of P450cam or Pdx.
Comparison with Previous Thermodynamic Studies--
The uptake of
water molecules would be supported by our recent thermodynamic studies
on the binding between ferric P450cam and Pdxox (16). Our
group has reported that the binding energetics of the ferric
P450cam·Pdxox complex is characterized by the negative
entropy change (
S =
93.2 J mol
1
K
1). It has been suggested that the decreased entropy is
partially caused by the ordering of water molecules in and around the
protein-protein interface. Xavier et al. (37) have estimated
the number of water molecules trapped in the association between
bobwhite quail lysozyme and the monoclonal antibody HyHEL-5 by using
the experimental data of the entropy changes of binding. Following
their method (37, 38), the entropy change of binding can be expressed
as the sum of the three types of entropy changes; the hydrophobic effect,
Shydr, the decrease in the rotational
and translational degrees of freedom,
Strans,
and the other contributions,
Sother as shown
in Equation 7.
|
(Eq. 7)
|
As discussed by Xavier et al. (37), the entrapment of
water molecules at the complex interface would be reflected by the term,
Sother.
Shydr
can be estimated by using the following relationship (39),
|
(Eq. 8)
|
where
Cp is the measured heat capacity
change. Studies in statistical mechanics and the experiments on
different protein-protein associations show that
Strans is approximately
209 J
mol
1 K
1 (40, 41). Because
S
and
Cp in the association of ferric P450cam
with Pdxox were determined as
93.2,
1290 J
mol
1 K
1, respectively (16),
Sother is estimated as about
364 J
mol
1 K
1. Dunitz (42) has used the entropy
of water in ice and hydrates of various salts to arrive at an upper
limit for the entropy of water tightly bound to the surface of a
protein as 29.3 J mol
1 K
1. Using this value
and assuming that all of
Sother is attributed to water uptake, an estimate of 12 trapped water molecules is obtained.
This number of the water molecules is in good agreement with that
obtained by the osmotic stress experiments in this paper (13 ± 2 water molecules). Both the osmotic pressure experiments and the
calorimetric studies, therefore, indicate the important contribution of
water molecules to the P450cam-Pdx interaction.
Role of the Interfacial Water Molecules in the P450cam·Pdx
Recognition--
So far, the uptake of water molecules has been
studied in the following systems; the binding of cytochrome
c by cytochrome c oxidase
(
VW = 224 cm3/mol) (34), the
binding of lysozyme with its antibody (
VW = 216 cm3/mol) (37), and several protein-DNA complexes (43).
For example, Bhat et al. (44) reported the 1.8 Å-resolution
crystal structure of the complex of hen egg white lysozyme (HEL) with
its antibody Fv D1.3, and found that the trapped water molecules at the
interface bridge two proteins by making hydrogen bonds with protein
residues. They also proposed that these water-mediated hydrogen bonds
contribute to the stabilization and the high specificity of the
antigen-antibody complex.
In the P450cam·Pdx complex, the formation of five direct interprotein
hydrogen bonds is proposed on the basis of the docking simulations
between P450cam and Pdx (9). If each hydrogen bond is mediated by 1 water molecule, only 5 water molecules could be explained among 25 or
13 water molecules obtained by osmotic stress. As seen in the case of
HEL/Fv D1.3, however, the water-mediated hydrogen bonds form the
extensive three-dimensional network that bridges two proteins. Whereas
two amino acids separated by 6 Å cannot form the hydrogen bond in
general, the water molecule at the intermediate position allows the
hydrogen bonding interaction between these two residues; amino acid
residue-(3.0 Å)-H2O-(3.0 Å)-amino acid residue. It might
therefore be possible that 25 (in P450cam·Pdxred) or 13 (in P450cam·Pdxox) water molecules are involved in the
hydrogen-bonding network to assist the protein-protein association.
Unfortunately, the crystal structure of P450cam·Pdx complex has not
yet been available, and we cannot assign the detailed location of the
trapped water molecules at the interface. Among many P450 proteins, the
complex structure between P450 and its partner protein has been
revealed only in P450BM-3 at 2.03 Å resolution (45). Between the heme
and the FMN domains of P450BM-3, there are two direct hydrogen bonds
and several water-mediated contacts. Sevrioukova et al. (45)
have also argued that well ordered water molecules at the interface
serve as hydrogen-bonded bridges between the two domains. Whereas the
redox partner is different between P450cam and the heme domain of
P450BM-3, the structure of P450cam is similar to that of the heme
domain of P450BM-3. It is, therefore, possible that the osmotically
labile water molecules are also trapped at the interface of
P450cam·Pdx complex to mediate the hydrogen-bonding interaction.
The buried water molecules at the interface are not necessarily
involved only in the hydrogen-bonding interactions. In the association
of bobwhite quail lysozyme with its antibody HyHEL-5 (37) and of
porphyrin-substituted cytochrome c with cytochrome c oxidase (34), several water molecules occupy the void at
the complex interface, which is caused by imperfect packing, resulting in the increase of the shape complementarity. Also in the crystal structure of P450BM-3 (Fig.
7A), there are significant
numbers of water molecules at the craggy domain-interface. Because of the unavailability of the P450cam·Pdx complex crystal structure, we
cannot currently give a definitive conclusion on the number of water
molecules involved in the hydrogen bond or in the increase of the shape
complementarity. However, the large number of osmotically labile waters
suggests that both of these effects may be responsible for the
P450cam-Pdx association. Indeed, as seen in the computer-simulated structure of the P450cam·Pdx complex (Ref. 9, Fig. 7B),
the complex interface is a craggy structure both on P450cam and Pdx, and the packing is not perfect. It can, therefore, be suggested that
water molecules fill the cavity at the P450cam·Pdx interface, increasing the shape complementarity and mediating the hydrogen-bonding interactions.

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|
Fig. 7.
Comparison of the crystal structure of
P450BM-3 with the computer-simulated structure of the P450cam·Pdx
complex. A, the crystal structure of the heme domain
(blue) complexed with the FMN domain (red) in
P450BM-3 (45). The water molecules detected in the crystal structure
are shown in green. B, computer-simulated
structure of the P450cam·Pdx complex, developed by Pochapsky et
al. (9). The atomic coordinates of this complex are a gift from
Prof. Pochapsky. The regions colored by red and
blue are Pdx and P450cam, respectively. The packing at the
complex interface is not perfect, and the cavity at the interface is
considered to be filled with water molecules.
|
|
It has been generally believed that for the formation of macromolecular
complexes, tighter binding is usually accompanied by a greater extent
of dehydration (13-15). In contrast, we find that high affinity
binding of ferric P450cam to Pdxred
(K
= 8.3 × 10
1 µM
1) is accompanied by
more water uptake (25 molecules) than the low affinity binding to
Pdxox (K
= 5.8 × 10
2 µM
1, uptake
of 13 water molecules). As discussed above, these water molecules are
considered to be involved in the formation of the hydrogen-bonding
network by filling the cavity at the interface. Therefore, one of the
possible explanations for the different
VW
between P450cam·Pdxred and P450cam·Pdxox is
that the hydrogen-bonding network at the interface would be more
extensive in the P450cam·Pdxred association than the
complex with Pdxox. In terms of the binding energetics, the
weaker association of K
than
K
corresponds to a free
energy difference of 6.5 kJ mol
1. Whereas the energetics
of the water-mediated hydrogen bonds is experimentally difficult to
examine, Williams et al. (46) have estimated that a buried
water-protein hydrogen bond stabilizes the folded protein by 2.5 kJ
mol
1. Langhorst et al. (47) have also studied
the water-mediated protein-protein interactions in RNase T1 and
reported that the free-energy contribution of the interactions of
Asn9 and Thr93 through water molecules to the
conformational stability does not exceed the absolute value of 1.3 kJ
mol
1. In comparison with the direct hydrogen bond (up to
about 12.6 kJ mol
1 stabilization, Ref. 48), the
water-mediated one will be weaker, but the interfacial water molecules
can both mediate noncomplementary donor-donor or acceptor-acceptor
pairs and connect nonoptimally oriented donor-acceptor pairs. It is,
therefore, possible that the redox-dependent affinity
difference of as much as 6.5 kJ mol
1 is manifested by the
rearrangement of about 10 interfacial water molecules as observed.
Actually, the structure of the P450cam·Pdxox and
P450cam·Pdxred complexes are believed to be different.
Although the structure of Pdx has been only determined in the oxidized
state, it has been reported that there are some structural changes upon
the reduction of Pdxox (4, 49-52). The 15N NMR
relaxation measurements have suggested that Pdxox exhibits
higher mobility than Pdxred, especially at residue
Asp34 forming the ionic pairs with Arg109 of
P450cam (49). As proposed by Pochapsky et al. (51), it is
also possible that upon the reduction of Pdxox the indole
ring of Trp106 would be partially inserted into a cleft on
the surface of the protein. Therefore, these
redox-dependent structural and/or dynamics changes in Pdx
would lead to a different structure at the complex interface between
ferric P450cam and Pdxox or Pdxred as
represented in Fig. 8, resulting in the
more extensive hydrogen-bonding network in the ferric
P450cam·Pdxred complex.

View larger version (12K):
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|
Fig. 8.
Schematic representation of the difference
between P450cam/Pdxred and P450cam/Pdxox
complexes. Interfacial water molecules are depicted as black
circles. Dotted lines are the plausible
hydrogen-bonding interactions. The larger number of interfacial water
molecules in the P450cam·Pdxred complex would reflect the
larger VW. The difference in the affinities
between complexes with Pdxox and Pdxred is due
to the number of the hydrogen-bonding interactions.
|
|
The molecular recognition by the water-mediated interactions discussed
above can also be seen in the restriction enzyme/DNA complex. Robinson
and Sligar (53) have suggested the participation of bound waters in the
sequence discrimination of substrate DNA by EcoRI by using
the osmotic pressure method. Whereas EcoRI binding occurs
more tightly at the recognition site (GAATTC) than the alternate DNA
sequence (TAATTC), there are ~70 water molecules in the
EcoRI-GAATTC complex that are not present in the complex with TAATTC. They have proposed that in the EcoRI system the
extent of bound water is critical to binding affinity,
sequence-specific recognition, and site discrimination during DNA
cleavage. As seen in EcoRI-DNA recognition, therefore,
ferric P450cam can also associate more tightly with Pdxred
through the extensive hydrogen-bonding network mediated by water molecules, which would be broken in the P450cam·Pdxox
complex, resulting in the higher affinity of P450cam with
Pdxred compared with Pdxox.
In summary, this study shows that water molecules can be one of the key
players in molecular recognition and probably in
redox-dependent affinity between P450cam and Pdx. It is
plausible that the extensive hydrogen-bonding network mediated by the
intervening water molecules contributes to the
redox-dependent affinity of P450cam against Pdx. To gain
more insight into the role of water molecules in P450cam-Pdx
association, we are currently investigating the dependence of the
association on hydrostatic pressure, which can also perturb the
hydration state of protein molecules.
 |
ACKNOWLEDGEMENT |
We are grateful to Prof. Yuzuru Ishimura (Keio
University), Prof. Tadao Horiuchi for a gift of the expression vector
of the P450cam and the Pdx genes, and Prof.
Thomas C. Pochapsky (Brandeis University) for a gift of the atomic
coordinates of the computer-simulated P450cam·Pdx structure. We are
also grateful to Dr. Koichiro Ishimori and Dr. Satoshi Takahashi for
fruitful discussions and Mr. Takehiko Tosha for experimental advice.
 |
FOOTNOTES |
*
This work was supported by a Grant-in-aid for Scientific
Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan 08249102 (to I. M.).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: Dept. of Molecular
Engineering, Graduate School of Engineering, Kyoto University, Kyoto
606-8501, Japan. Tel.: 81-75-753-5921; Fax: 81-75-751-7611; E-mail:
morisima@mds.moleng.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M010217200
2
A small amount of P450cam-CO sometimes exists
before the laser experiment partially because of the room light. Such
preformed P450cam-CO is converted to the ferrous form by the laser
irradiation. To eliminate the small contribution by the absorbance
change from P450cam-CO to ferrous P450cam, the measurements for the
Pdx redox process were done at the isosbestic point, 467.4 nm, between
ferrous P450cam and P450cam-CO.
3
We also observed the kinetics of ferric P450cam
by monitoring the absorbance change at 650 nm. The ferric form of
P450cam has a charge-transfer band at 650 nm, whereas the ferrous and CO-form of P450cam do not. In all conditions used in this study, a
laser pulse decreases the absorption at 650 nm with the same rate
constant as that obtained from the absorbance change at 446 nm. As
discussed by Hintz et al. (21), this is consistent with the expectation
that the CO-binding process is not the rate-determining step under our
experimental conditions.
 |
ABBREVIATIONS |
The abbreviations used are:
P450cam, cytochrome P450 camphor;
PdR, putidaredoxin reductase;
Pdx, putidaredoxin;
ET, electron transfer;
MV, methyl viologen.
 |
REFERENCES |
1.
|
Mueller, E. J.,
Loida, P. J.,
and Sligar, S. G.
(1995)
in
Cytochrome P450, Structure, Mechanism, and Biochemistry
(Oritz de Montellano, P. R., ed)
, pp. 83-124, Plenum Press, New York
|
2.
|
Davies, M. D.,
and Sligar, S. G.
(1992)
Biochemistry
31,
11383-11389[Medline]
[Order article via Infotrieve]
|
3.
|
Lipscomb, J. D.,
Sligar, S. G.,
Namtvedt, M. J.,
and Gunsalus, I. C.
(1976)
J. Biol. Chem.
251,
1116-1124[Abstract]
|
4.
|
Stayton, P. S.,
and Sligar, S. G.
(1991)
Biochemistry
30,
1845-1851[Medline]
[Order article via Infotrieve]
|
5.
|
Stayton, P. S.,
and Sligar, S. G.
(1990)
Biochemistry
29,
7381-7386[Medline]
[Order article via Infotrieve]
|
6.
|
Unno, M.,
Shimada, H.,
Toba, Y.,
Makino, R.,
and Ishimura, Y.
(1996)
J. Biol. Chem.
271,
17869-17874[Abstract/Free Full Text]
|
7.
|
Holden, M.,
Mayhew, M.,
Bunk, D.,
Roitberg, A.,
and Vilker, V.
(1997)
J. Biol. Chem.
272,
21720-21725[Abstract/Free Full Text]
|
8.
|
Aoki, M.,
Ishimori, K.,
and Morishima, I.
(1998)
Biochim. Biophys. Acta
1386,
157-167[Medline]
[Order article via Infotrieve]
|
9.
|
Pochapsky, T. C.,
Lyons, T. A.,
Kazanis, S.,
Arakaki, T.,
and Ratnaswamy, G.
(1996)
Biochimie (Paris)
78,
723-733[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Roitberg, A. E.,
Holden, M. J.,
Mayhew, M. P.,
Kurnikov, I. V.,
Beratan, D. N.,
and Vilker, V. L.
(1998)
J. Am. Chem. Soc.
120,
8927-8932[CrossRef]
|
11.
|
Sligar, S. G.,
Debrunner, P. G.,
Lipscomb, J. D.,
Namtvedt, M. J.,
and Gunsalus, I. C.
(1974)
Proc. Natl. Acad. Sci. U. S. A.
71,
3906-3910[Abstract]
|
12.
|
Jelesarov, I.,
and Bosshard, H. R.
(1994)
Biochemistry
33,
13321-13328[Medline]
[Order article via Infotrieve]
|
13.
|
Bogan, A. A.,
and Thorn, K. S.
(1998)
J. Mol. Biol.
280,
1-9[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Conte, L. L.,
Chothia, C.,
and Janin, J.
(1999)
J. Mol. Biol.
285,
2177-2198[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Chothia, C.,
and Janin, J.
(1975)
Nature
256,
705-708[Medline]
[Order article via Infotrieve]
|
16.
|
Aoki, M.,
Ishimori, K.,
Fukuda, H.,
Takahashi, K.,
and Morishima, I.
(1998)
Biochim. Biophys. Acta
1384,
180-188[Medline]
[Order article via Infotrieve]
|
17.
|
Parsegian, V. A.,
Rand, R. P.,
and Rau, D. C.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3987-3992[Abstract/Free Full Text]
|
18.
|
Di Primo, C. C.,
Sligar, S. G.,
Hui Bon Hoa, G.,
and Douzou, P.
(1992)
FEBS Lett.
312,
252-254[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Gunsalus, I. C.,
and Wagner, G. C.
(1978)
Methods Enzymol.
52,
166-188[Medline]
[Order article via Infotrieve]
|
20.
|
Davies, M. D.,
Qin, L.,
Beck, J. L.,
Suslick, K. S.,
Koga, H.,
Horiuchi, T.,
and Sligar, S. G.
(1990)
J. Am. Chem. Soc.
112,
7396-7398
|
21.
|
Hintz, M. J.,
and Peterson, J. A.
(1981)
J. Biol. Chem.
256,
6721-6728[Free Full Text]
|
22.
|
Hintz, M. J.,
Mock, D. M.,
Peterson, L. L.,
Tuttle, K.,
and Peterson, J. A.
(1982)
J. Biol. Chem.
257,
14324-14332[Free Full Text]
|
23.
|
Kido, T.,
and Kimura, T.
(1979)
J. Biol. Chem.
254,
11806-11815[Abstract]
|
24.
|
Weast, R. C.
(1977)
CRC Handbook of Chemistry and Physics
, 58th Ed.
, pp. D228-D231, CRC Press, Inc., Cleveland
|
25.
|
Rand, R. P.,
Fuller, N. L.,
Butko, P.,
Francis, G.,
and Nicholls, P.
(1993)
Biochemistry
32,
5925-5929[Medline]
[Order article via Infotrieve]
|
26.
|
Furukawa, Y.,
Ishimori, K.,
and Morishima, I.
(2000)
Biochemistry
39,
10996-11004[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Unno, M.,
Christian, J. F.,
Benson, D. E.,
Gerber, N. C.,
Sligar, S. G.,
and Champion, P. M.
(1997)
J. Am. Chem. Soc.
119,
6614-6620[CrossRef]
|
28.
|
Aoki, M.,
Ishimori, K.,
Morishima, I.,
and Wada, Y.
(1998)
Inorg. Chim. Acta
272,
80-88[CrossRef]
|
29.
|
Koga, H.,
Sagara, Y.,
Yaoi, T.,
Tsujimura, M.,
Nakamura, K.,
Sekimizu, K.,
Makino, R.,
Shimada, H.,
Ishimura, Y.,
Yura, K.,
Go, M.,
Ikeguchi, M.,
and Horiuchi, T.
(1993)
FEBS Lett.
331,
109-113[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Di Primo, C.,
Deprez, E.,
Hui Bon Hoa, G.,
and Douzou, P.
(1995)
Biophys. J.
68,
2056-2061[Abstract]
|
31.
|
Harris, D.,
and Loew, G.
(1993)
J. Am. Chem. Soc.
115,
8775-8779
|
32.
|
Marcus, R. A.,
and Sutin, N.
(1985)
Biochim. Biophys. Acta
811,
265-322
|
33.
|
Kornblatt, M. J.,
Kornblatt, J. A.,
and Hui Bon Hoa, G.
(1993)
Arch. Biochem. Biophys.
306,
495-500[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Kornblatt, J. A.,
Kornblatt, M. J.,
Hui Bon Hoa, G.,
and Mauk, A. G.
(1993)
Biophys. J.
65,
1059-1065[Abstract]
|
35.
|
Mauk, M. R.,
Reid, L. S.,
and Mauk, A. G.
(1982)
Biochemistry
21,
1843-1846[Medline]
[Order article via Infotrieve]
|
36.
|
Kresheck, G. C.,
Vitello, L. B.,
and Erman, J. E.
(1995)
Biochemistry
34,
8398-8405[Medline]
[Order article via Infotrieve]
|
37.
|
Xavier, K. A.,
Shick, K. A.,
Smith-Grill, S. J.,
and Wilson, R. C.
(1997)
Biophys. J.
73,
2116-2125[Abstract]
|
38.
|
Kondo, H.,
Shiroishi, M.,
Matsushima, M.,
Tsumoto, K.,
and Kumagai, I.
(1999)
J. Biol. Chem.
274,
27623-27631[Abstract/Free Full Text]
|
39.
|
Spolar, R. S.,
and Record, M. T., Jr.
(1994)
Science
263,
777-784[Medline]
[Order article via Infotrieve]
|
40.
|
Janin, J.,
and Chothia, C.
(1978)
Biochemistry
17,
2943-2948[Medline]
[Order article via Infotrieve]
|
41.
|
Finkelstein, A. V.,
and Janin, J.
(1989)
Protein Eng.
3,
1-3[Medline]
[Order article via Infotrieve]
|
42.
|
Dunitz, J. D.
(1994)
Science
264,
670
|
43.
|
Robinson, C. R.,
and Sligar, S. G.
(1996)
Protein Sci.
5,
2119-2124[Abstract/Free Full Text]
|
44.
|
Bhat, T. N.,
Bentley, G. A.,
Boulot, G.,
Greene, M. I.,
Tello, D.,
Dall'Acqua, W. D.,
Souchon, H.,
Schwarz, F. P.,
Mariuzza, R. A.,
and Poljak, R. J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1089-1093[Abstract]
|
45.
|
Sevrioukova, I. F.,
Li, H.,
Zhang, H.,
Peterson, J. A.,
and Poulos, T. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1863-1868[Abstract/Free Full Text]
|
46.
|
Williams, M. A.,
Goodfellow, J. M.,
and Thornton, J. M.
(1994)
Protein Sci.
3,
1224-1235[Abstract/Free Full Text]
|
47.
|
Langhorst, U.,
Backmann, J.,
Loris, R.,
and Steyaert, J.
(2000)
Biochemistry
39,
6586-6593[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Fersht, A. R.,
Shi, J. P.,
Knill-Jones, J.,
Lowe, D. M.,
Wilkinson, A. J.,
Blow, D. M.,
Brick, P.,
Carter, P.,
Waye, M. M.,
and Winter, G.
(1985)
Nature
314,
235-238[Medline]
[Order article via Infotrieve]
|
49.
|
Sari, N.,
Holden, M. J.,
Mayhew, M. P.,
Vilker, V. L.,
and Coxon, B.
(1999)
Biochemistry
38,
9862-9871[CrossRef][Medline]
[Order article via Infotrieve]
|
50.
|
Pochapsky, T. C.,
Jain, N. U.,
Kuti, M.,
Lyons, T. A.,
and Heymont, J.
(1999)
Biochemistry
38,
4681-4690[CrossRef][Medline]
[Order article via Infotrieve]
|
51.
|
Pochapsky, T. C.,
Ratnaswamy, G.,
and Patera, A.
(1994)
Biochemistry
33,
6433-6441[Medline]
[Order article via Infotrieve]
|
52.
|
Roitberg, A. E.
(1997)
Biophys. J.
73,
2138-2148[Abstract]
|
53.
|
Robinson, C. R.,
and Sligar, S. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2186-2191[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.