From the
Using the fluorescent probe ADIFAB (acrylodan-derivatized
intestinal fatty acid-binding protein) to determine the equilibrium
concentration of the free (unbound) fatty acid (FFA), dissociation
constants were measured between 10 and 50 °C for the interaction of
five different long chain fatty acids (FA) with fatty acid-binding
proteins (FABP) from adipocyte, intestine, and heart. Gibbs free
energies (
Fatty acid-binding proteins (FABP)
As discussed
previously(12) , the uniformity and magnitude of binding
affinities are surprising for several reasons. 1) FABPs from different
tissues exhibit considerable differences in amino acid
sequence(6) . 2) The conformation of the bound ligand as
revealed by x-ray crystallography differs with FABP type and to some
extent with FA type (6, 7, 14). 3) Different FA have very different
aqueous solubilities (12, 15). 4) Rates of transfer of the anthroyloxy
FA from FABP to membranes are quite different for different
FABPs(16) . 5) FABPs with K
We have recently reinvestigated the
binding of FA to FABP using a fluorescent probe, composed of
acrylodan-derivatized intestinal FABP and denoted ADIFAB(12) .
In contrast to most previous methods for estimating binding affinities,
the ADIFAB method allows one to determine the concentration of FFA in
equilibrium with FABP without separating the reaction products. The
equilibrium dissociation constants (K
This ability to measure accurately the binding affinities
makes it possible to investigate the specific interactions that
contribute to the free energies of binding. Interactions that are
likely to contribute significantly include those, as indicated by x-ray
crystallography, that involve the interaction of the FA carboxylate
through a network of hydrogen bonds with specific Arg residues in the
FA binding cavity of the FABP(9, 18, 19) . X-ray
crystallography also reveals that large numbers of both ordered and
disordered water molecules within the FA binding cavity are displaced
upon FA binding, and this might also contribute to the free energy of
binding. In addition to these interactions that involve the FABP
directly, our previous results from equilibrium binding measurements
using ADIFAB at 37 °C indicate that FA solubility in water plays an
important role in determining the relative binding affinities of
different types of FA (12). Moreover, these binding measurements also
suggest that the magnitude of the free energy change upon transferring
a FA from the bulk solvent to the FABP cavity depends on the overall
hydrophobicity of the amino acids within the cavity.
In general, the
binding of FA to FABP can be described in terms of several discrete
events, each of which contribute to the overall free energy. The free
energy corresponding to the observed equilibrium constants can be
represented as a sum of the difference of free energies of the two
states defined as: 1) FA in water + FABP without FA (apoFABP) and
2) water without FA + FA bound to FABP (holoFABP). Thus, the
change in the free energy of binding should be a sum of contributions
from the changes in the free energy of 1) the bulk solvent, 2) the FA,
3) the protein, and 4) the protein-bound water. In the present study
each of these contributions has been estimated using both experimental
and theoretical information. The change in the free energy, enthalpy,
and entropy of FA binding to FABP was determined by measuring binding
at temperatures between 10 and 50 °C. The temperature dependence of
the partition of FA between membranes and water was also measured, and
these values were used to estimate the change in thermodynamic
parameters of bulk solvent upon removal of FA. These results help to
elucidate the nature of the binding interaction between FA and FABP.
On-line formulae not verified for accuracy and
On-line formulae not verified for accuracy where R is the measured ratio of 505 to 432 nm intensities
(with blank intensities subtracted), R
The concentration of bound
FABP can be expressed in terms obtained from ADIFAB fluorescence
(Equations 1 and 2) as shown by Equation
3.
On-line formulae not verified for accuracy Using Equations 1-3, a single-site Scatchard analysis was
done in which, for each binding isotherm, the fraction (
On-line formulae not verified for accuracy and this quantity, divided by [FFA], was fitted
to
On-line formulae not verified for accuracy in which n is the stoichiometry and K
On-line formulae not verified for accuracy where V
Miyazaki et al.(26) and
Peitzsch and McLaughlin (27) have found that the membrane/water
partition coefficients for the uncharged FA (FAH) are about 200-fold
greater than for the charged species (FA
Free energies for the dissociation of FA from
FABP were evaluated from Equation
8.
On-line formulae not verified for accuracy The corresponding enthalpy changes were determined from the slope
of van't Hoff plots and entropy changes were determined from
The dissociation constants obtained from this analysis, which are
displayed as van't Hoff plots in Fig. 1, illustrate that
the affinities for ADIFAB decrease with increasing temperature for all
FA. Fig. 1also shows that the van't Hoff plots are linear,
and fits to these plots were used to obtain the thermodynamic
parameters for FA binding to ADIFAB as shown in . These
results show that -T
In the present studies we have measured the temperature
dependence of the binding of fatty acids to FABPs from adipocyte,
intestine, and heart, to probe the chemical nature of the interactions
between FA and FABP. Thermodynamic parameters determined from these
measurements show that, with the exception of ADIFAB, the free energy
of binding is almost totally dominated by enthalpic contributions; for
ADIFAB the entropic contribution dominates. In contrast to the
substantial temperature dependence of FA binding to FABPs, partition of
these same FA into membranes is independent of temperature, suggesting
that membrane partition is dominated by entropic contributions.
However, the dependence on FA type is similar for both FABP and
membranes; binding affinities and K
The
significantly larger entropy increase upon FA binding to ADIFAB, as
compared to I-FABP, is consistent with an increase in rotational
freedom of the acrylodan moiety in the holo- as compared to the
apoADIFAB(15) . Evidence for this increase is the decrease in
fluorescence polarization of acrylodan from 0.32 in apoADIFAB to 0.15
in holoADIFAB(15) . While the value 0.15 is consistent with
considerable local rotational mobility, the value 0.3 is that expected
for rotation of the 15-kDa protein as a whole and therefore consistent
with virtually no local rotational mobility of the acrylodan moiety. In
addition, molecular modeling of the apo and holo crystal structures
suggests that the orientation of Lys
Consistent with our previous measurements of the K
In addition to these general trends, the results of I
reveal significant differences among the different FABPs and for a
given FABP significant differences among the different FA. For example,
although binding of palmitate to I-FABP has virtually no entropic
contribution, binding to heart, and especially adipocyte reveal
substantial entropic and correspondingly much smaller enthalpic
contributions. These results suggest that the palmitate conformation is
less constrained within the adipocyte and heart FABP binding cavities
and that specific interactions between the FA and the amino acids and
bound water within the binding cavities are substantially greater in
the intestinal FABP. (To ensure that the adipocyte results were not
idiosyncratic for the human form of the protein, measurements were also
done using the murine form and virtually identical results were
obtained as summarized in I, part a.) Moreover, within the
adipocyte binding cavity, palmitate and oleate display greater
differences in their thermodynamic parameters than in heart and
intestine. The almost 4 kcal/mol greater enthalpic contribution for
binding of oleate to adipocyte, as compared to palmitate, probably has
its origin in different interactions between the hydrocarbon portions
of the FA and the amino acid side chains and/or bound water
molecules(19) . Indeed, analysis of the crystal structure
reveals that oleate and palmitate have similar carboxylate
conformations within the adipocyte binding cavity, but palmitate has
substantially greater numbers of non-polar amino acid contacts along
its hydrocarbon chain than does oleate(11, 19) .
Furthermore, because the average temperature coefficient of stearate is
substantially greater than that of oleate(11) , the greater
conformational flexibility suggested for palmitate may be
characteristic of saturated FA.
The observed thermodynamic characteristics of FA binding to FABPs
and partition into membranes reflect the sum of contributions of
several interactions involving the FABP, membrane, FA, and water. To
assess the relative importance of each of the interactions that govern
FA binding to FABP, we will, in the following discussion, estimate the
magnitude of the individual interactions involved in FA binding to FABP
using the measured binding results as well as estimates obtained from
theory and model systems. We begin by expressing the free energy of
binding or, equivalently, the free energy for transfer of FA from water
to FABP (
On-line formulae not verified for accuracy
The term
On-line formulae not verified for accuracy
The second contribution to
To compare the measured and estimated thermodynamic
parameters, we adopt the following values as representative of the
measured values of I:
The
reliability of these conclusions rests on the assumptions made to
obtain the estimates for the individual terms in . In
particular, the contributions due to displacement of bound water may
not be negligible. This displacement might involve favorable entropy
changes that would help balance the FA conformational and any
additional unfavorable entropic contributions embodied in the SI term.
The displacement of bound water is unlikely, however, to contribute
negative enthalpy changes, needed to balance +5 kcal/mol
contribution from
The
results presented here suggest that several large thermodynamic terms
that characterize FA binding to FABP tend to cancel. The net entropy
portion is approximately zero because the large favorable entropy
change of the solvent is canceled by an equally large but unfavorable
entropy change, most likely due to restraints on the FA configuration.
These entropy changes are to a large extent independent of the FABP,
and therefore interactions that are FABP-specific are predominantly
enthalpic. The large net (
Although
overall enthalpy dominates the binding energy, relatively small
entropic contributions play significant roles in determining
differences in binding affinities for different FA. As emphasized
previously, FA solubility, which contributes to the binding interaction
as entropy, plays a major role in determining the differences in FA
binding to a given FABP(12) . As seen in I, net
entropy differences for different FA are at least 2 kcal/mol, and this
energy difference corresponds to an affinity difference of about
30-fold. Thus, while net enthalpy may contribute the bulk of the net
free energy, even relatively small variations in the net entropy
contributions can have significant effects on binding affinities.
All values are in kcal/mol,
and
Units and
abbreviations are listed in Table I.
Units and abbreviations are listed in Table
I.
This table compares measured
thermodynamic parameters that are representative of all FA and all
FABPs (bottom row) with the sum of terms (row labeled Total) that
appear in equation (9). For each term the values of
We thank Dr. Marvin Rich for making available results
of studies before publication and Dr. Judith Storch for helpful
comments about the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
G) determined from the dissociation constants
were between about -9 and -11 kcal/mol at 25 °C.
Thermodynamic parameters for binding were determined using van't
Hoff plots of the dissociation constants, which range, over the entire
temperature region, between 2 and 3000 nM. For all the
unlabeled FABPs, free energies of binding were dominated by large
negative enthalpies that ranged from -7 to -12 kcal/mol,
and the enthalpies tended to decrease with increasing FA unsaturation.
The entropic contributions (-T
S) at 25
°C ranged between -4 and +2 kcal/mol and tended to
increase with increasing FA unsaturation. To assess the role of FA
aqueous solubility in FABP binding, measurements of the partition of FA
between unilamellar lipid vesicles and water were also done using
ADIFAB; the lipid/water partition coefficients (K
)
determined from these measurements were found to be independent of
temperature. The binding of FA to FABP is governed by the sum of
contributions of various interactions between FA, water, and FABP. An
analysis of the individual contributions suggests that the net free
energy of binding results from the canceling in part of a number of
separate quite large contributions. The entropic contributions sum
almost to zero for most FA and FABPs as a result of the canceling of a
large increase in bulk solvent entropy by decreases in configurational
entropy upon FA binding to FABP. The net, approximately -10
kcal/mol enthalpy of binding, probably results from an increase in FA
configurational enthalpy upon binding to FABP plus a large negative
enthalpy from the interaction between the FA and the FABP. This large
enthalpy of the FA-FABP interaction suggests that in addition to
previously identified specific interactions between the carboxylate
portion of the FA and charged amino acids within the binding cavity,
other significantly larger enthalpic interactions, presumably involving
the hydrocarbon portion of the FA, must contribute to the binding
energy.
(
)are
14-15-kDa proteins found in the cytosols of a variety of tissues
(1-8). Considerable binding and structural evidence indicates
that fatty acids (FA) are the natural ligands for these
proteins(9, 10, 11, 12) . Most studies
of the binding of FA to FABP have revealed relatively little variation
in affinity either as a function of the type of FA or FABP and are
generally consistent with a K
of about 1
µM (Ref. 13; see reviews in Refs. 6 and 12). These
previous results, therefore, suggested that the interactions that
govern binding are similar for all FA and all FABPs.
values
on the order of 1 µM would not be expected to interact
appreciably with FA under physiologic conditions because serum FFA is
about 7 nM(17) .
)
measured at 37 °C by this method range, for different FABPs and
different FA, between 2 and 1000 nM, indicating much greater
binding affinities as well as greater heterogeneity than previous
studies.
Materials
Sodium salts of FA were purchased from
NuChek Prep, Elysian, MN. Stock solutions of FA were prepared at
20-50 mM in water with 25 µM butylated
hydroxytoluene, pH 9.0, and stored under argon at -20
°C. The buffer used to measure FA binding to FABPs consisted of 10
mM HEPES, 150 mM NaCl, 5 mM KCl, and 1
mM NaHPO
, at pH 7.4. The pET11a and pET11d
expression vectors and the BL21 (DE3) Escherichia coli strain
were purchased from Novagen, Madison, WI. Lipidex-5000 was purchased
from Packard Instruments and Sigma. The fluorescent I-FABP, ADIFAB, was
prepared from acrylodan-derivatized recombinant rat intestinal fatty
acid-binding protein (rI-FABP) as described (15) and is
available from Molecular Probes, Eugene, OR.
Lipid Vesicles
Large unilamellar vesicles composed
of egg phosphatidylcholine were prepared by extruding the hydrated
lipid 10 times through two stacked 0.1-µm polycarbonate filters
(Nucleopore) at room temperature, essentially as
described(20, 21) . Lipid was dissolved in chloroform,
evaporated to dryness, and lyophilized overnight, and the dried lipid
film was hydrated in buffer.
Preparation of FABP Protein
All FABPs (rat
intestine, human and mouse adipocyte, and rat heart) were recombinant
proteins that were expressed in the BL21 (DE3)/pET11 host/vector
expression system as described previously(12, 15) . All
of the recombinant FABPs were purified from cell lysates by a
modification of the method of Lowe et al.(22) , as
described previously(12, 15) . All FABP protein was
delipidated to remove fatty acids by slow (3-5 h) Lipidex-5000
chromatography at 37 °C, and FABP purity was assessed by SDS and
isoelectric focusing polyacrylamide gel electrophoresis. FABP
concentration was determined principally by UV absorbance using the
consensus protein molar extinction coefficients(12) .
[FFA] Determination with ADIFAB
ADIFAB
responds to FA binding by undergoing a shift in fluorescence emission
from 432 nm in the apo, to 505 nm in the holo form(15) . As
described previously(12, 15, 21) , the
concentrations of FFA and FA bound to ADIFAB can be determined
according to, is this
ratio with no FFA present, and K
is the
equilibrium constant. The concentration of FFA in equilibrium with
ADIFAB was computed as the total FA added to the cuvette minus the
amount bound to ADIFAB minus the amount bound to the cuvette walls. The
amount bound to the walls, for each FA and at temperatures between 10
and 50 °C, was determined by using ADIFAB to measure the change in
[FFA] upon transfer of the sample between cuvettes as
described in Ref. 15. Binding of polyunsaturated FA was
temperature-independent and less than 14%. For palmitate and oleate,
wall binding was as much as 35%; an amount that decreased with
decreasing temperature. Measurements were done either with an SLM 8000C
or 4800 fluorometer, and standard deviations of the measured R values were typically <0.3%.
Measurement of FA-FABP Equilibrium Binding
The
method used to determine FA binding affinities for FABP has been
described previously(12) . Briefly, the binding of FA to a given
FABP was determined by using ADIFAB, in the presence of fixed
concentrations of FABP, to monitor FFA levels in equilibrium with
ADIFAB and FABP. Thus FA was added in discrete aliquots to a mixture of
ADIFAB and FABP, and after each aliquot the ADIFAB fluorescence was
measured to determine FFA and ADIFAB. FA titrations were
done with an ADIFAB concentration of 0.2 µM (3 µg/ml)
and FABP concentrations between 1 and 20 µM. Each aliquot
of FA, allowing about 10 min for equilibration between aliquots, was
added to the cuvette from FA stocks warmed to temperatures above the FA
phase transition, and mixed thoroughly. Although a fraction,
proportional to [FFA], of the added FA binds to the walls of
the cuvette(15) , this value was negligible in the presence of
FABP or membranes at the concentrations used in the binding or
partition measurements, respectively.
) of FABP
that has FA bound relative to the total FABP was determined
as
the dissociation constant(23) . Experimental values
of
/[FFA] were fitted to Equation 5 using a weighted
linear regression with standard deviations of about 5% (Origin,
MicroCal Software, Northampton, MA).
Determination of Equilibrium Constants from Binding
Kinetics
Equilibrium constants for FA binding to ADIFAB were
also determined using the on and off rate constants (K = k
/k
) measured by
stopped-flow fluorescence as described previously(12) . An SLM
Milliflow stopped-flow device coupled to an SLM fluorometer was used to
measure the time course of the 505/432 R value. The off rate
constant was measured by mixing FA-ADIFAB complexes with fatty
acid-free bovine serum albumin, and the on rate was measured by mixing
FA and ADIFAB.
Partition Coefficients
Partition coefficients were
measured as described previously(24) . The coefficient
describing partition of FA between membrane and aqueous phase is
defined as: K =
[FA]
/[FFA], where [FA]
is the concentration of FA in the membrane phase. Expressing
these concentrations in terms of the total sample
volume,
and V
are the
volumes of the aqueous and membrane phases, respectively, and
[FA
] is the concentration of membrane-bound FA
relative to the total sample volume (
V
). In
this equation the value of V
/V
for lipid vesicles was estimated as 10
per
molar phospholipid (V
/V
= [L], where [L] is the unit-less
value of the molar lipid concentration), using an area per lipid
molecule of 70 Å
and a bilayer width of 40
Å(25) .
). The
partition coefficient determined using Equation 6 is a combination of
the partition coefficients for FAH and FA
. To
determine the thermodynamic parameters that apply to a single species,
the partition coefficients for individual species must be evaluated
from the measured K
values. To do this, we follow
Peitzsch and McLaughlin (27) and note that K
of Equation 6 can be expressed in terms of the single species
partition coefficients as K
=
K
Thermodynamic Parameters
To obtain the
thermodynamic parameters for the partitioning of FA into membranes, we
followed the approach of Peitzsch and McLaughlin(27) , who
expressed the free energy for transfer of the FA from water into
membranes (G
) in terms of the mole
fraction ratio X
/X
. The mole
fraction of FA within the membrane is X
[FA
]/[L], where
[FA
] and [L] are the concentration in
the total sample volume of FA and lipid, respectively. Similarly, the
mole fraction of FA within the aqueous phase is X
[FFA]/55.6. In terms of the partition coefficient
of the charged species of FA (K
G =
H - T
S.
Temperature Dependence of FA Binding to
ADIFAB
Because the probe ADIFAB was used to measure FA binding
to unlabeled FABPs and membranes as a function of temperature, it was
necessary first to determine the temperature dependence of binding to
ADIFAB. This was done for five different FA (palmitate,
oleate, linoleate, linolenate, and arachidonate) at temperatures
between 10 and 50 °C using the same methods described previously
for studies done at 37 °C (Ref. 15 and ``Experimental
Procedures''). Binding isotherms were determined at each
temperature and for each FA. Each isotherm was analyzed by single-site
binding to determine the dissociation constant at each temperature.
S increases, from
-6 to -3.7 kcal/mol, and
H decreases, from
about -2.9 to -4.4 kcal/mol, with double bond number.
Figure 1:
Temperature dependence of the binding
of FA to ADIFAB. The opensymbols in these
van't Hoff plots represent K values for each FA (see
legend of Table I for FA code) determined by the equilibrium
measurements described by Equations 1-5 under ``Experimental
Procedures,'' and the lines drawn through these symbols
are the linear fits to these data, whose slopes were used to determine
H.
Dissociation constants were also determined kinetically for selected
temperatures and FA using the methods described
previously(12, 15) . On rate constants were determined
by measuring the increase in R value as FA binds to ADIFAB,
following the stopped-flow mixing of apoADIFAB and FA. Off rate
constants were determined by measuring the decrease in R value
as FA dissociates from ADIFAB following the stopped-flow mixing of
holoADIFAB with fatty acid-free albumin. Measured off and on rate
constants for oleate, together with dissociation constants calculated
from these kinetic measurements, are listed in . Also
listed in are the corresponding K values obtained from the linear fit to the equilibrium
binding measurements of Fig. 1. As is apparent, dissociation
constants determined from equilibrium binding and kinetic measurements
are in excellent agreement.
Temperature Dependence of FA Binding to Native
FABPs
Binding isotherms were measured for FABPs from human
adipocyte (A-FABP), rat intestine (I-FABP), and rat heart (H-FABP),
using ADIFAB to measure the FFA concentration. Each isotherm was
analyzed using a single-site binding model to determine binding
constants and stoichiometries for each of five different FA, at
temperatures between 10 and 50 °C. The results of these
measurements are shown as van't Hoff plots of the measured K values in Fig. 2and indicate, as
in the case of ADIFAB, that for all native FABPs and all FA, the
binding affinities decrease with increasing temperature.
H were determined from the van't Hoff plots, and these values,
together with
G and -T
S, are
listed in I, parts a-c. These results indicate that
in most cases binding enthalpies are between -9 and -11
kcal/mol, and the -T
S values are generally
near zero. As in the case of ADIFAB, there is a general trend of
increasing -T
S and decreasing
H with increasing degree of FA unsaturation for all FABPs.
Figure 2:
Temperature dependence of the binding of
FA to FABP. The symbols in these van't Hoff plots represent K values for each FA (see legend of Table I for FA code) determined
by the equilibrium measurements described by Equations 1-5 under
``Experimental Procedures.'' A, human adipocyte
FABP; B, rat heart FABP; C, rat intestine
FABP.
Binding stoichiometries for all FA and FABPs were found to be
invariant with temperature (data not shown). As found previously at 37
°C, the stoichiometries were consistent with unity for the
adipocyte and intestinal proteins but were significantly less than
unity for the heart FABP(12) . Specifically, stoichiometries
averaged over all temperatures and FA were: 0.90 ± 0.09 for
adipocyte, 0.79 ± 0.14 for intestine, and 0.57 ± 0.07 for
the heart FABP. As discussed previously, we could find no evidence for
loss or degradation of protein that might account for the low
stoichiometry value of the heart (muscle) proteins (these low values
previously were observed for both bovine and rat heart). Nor could we
obtain definitive evidence for dimer formation, which might reduce
accessibility to the binding site if the interaction surfaces between
the dimers involved the portal region of the protein. Recently, the
x-ray crystal structure of FABP has been obtained from desert locust Schistocerca gregaria (8). This protein is also a muscle FABP,
and its structure reveals a symmetric dimer in which the contact
surfaces involve the region analogous to the presumed portal region of
the heart protein(35) . Thus, the muscle protein may be in the
form of a portal to portal dimer, which limits binding to only one of
the two sites/dimer, but for reasons unknown dimers of the rat heart
FABP, which contains no cysteine residues, cannot be detected by
molecular sizing chromatography(12) . (Dimers mediated by
disulfide linkages can of course be so detected, as found recently for
the porcine heart FABP (36).)
Temperature Dependence of Membrane Partition of
FA
The change in free energy upon binding of FA to FABP must
involve energy changes in the bulk solvent as well as the FABP. To
assess the aqueous contribution to the free energy change, measurements
were done to determine the temperature dependence of partition of FA
between water and lipid bilayer membranes. The unilamellar lipid
bilayer vesicles used in these measurements were prepared from egg
phosphatidylcholine by the extrusion method. Membranes were used for
these studies because they approximate a simple hydrophobic phase and
because values of the partition coefficient (K)
are relatively insensitive to membrane composition and/or physical
state(21, 27) . Partition coefficients were determined
by using ADIFAB to measure [FFA] in equilibrium with these
vesicles as described under ``Experimental Procedures'' and
previously(21) . Thus, FA were added to mixtures of ADIFAB (0.2
µM) and lipid vesicles (100 µM in
phospholipid), and the measured R values were used to
determine [FFA] for each FA, at temperatures between 10 and
50 °C. For these vesicle concentrations, most of the FA is in the
membrane, a small fraction binds to ADIFAB, and the rest (FFA) is in
the water (under the conditions of these studies, [FFA] is
well below the concentration at which FA aggregation occurs). K
values were determined from the measured
[FFA] using Equation 6 and converted to mole fractions as
described under ``Experimental Procedures.'' These results
are displayed as van't Hoff plots in Fig. 3. They show that
for all FA, K
values are virtually invariant with
temperature; therefore, as indicated in the figure legend, no
significant net enthalpy is involved in the water-membrane transition.
The variation of K
with FA type, as discussed
previously(21) , is largely governed by the relative solubility
of the different FA; the least soluble saturated FA have the largest K
.
Figure 3:
Temperature dependence of the partition of
FA into lipid vesicles. The membrane/water mole fractions (X/X
) for each FA were
determined from the measured partition coefficients as described under
``Experimental Procedures'' (see legend of Table I for FA
code). Enthalpies (kcal/mol) for each of the FA are: palmitate (PA, 16:0), -0.4 ± 0.8; oleate (OA,
18:1), 1.1 ± 0.7; linoleate (LA, 18:2), 0.5 ±
0.8; linolenate (LNA, 18:3), -0.3 ± 0.7;
arachidonate (AA, 20:4), 1.1 ±
0.7.
values
decrease with increasing numbers of double bonds at all temperatures.
In the following we will first discuss the specific thermodynamic
features and binding characteristics of the individual FABPs and will
then address the issue of what interactions in general may be
responsible for the overall thermodynamics of FA binding to FABPs.
ADIFAB
Because the measurements in this study were done
using ADIFAB, the validity of the results for all FABPs relies on the
accuracy of the K values for ADIFAB. The
fact that K
values determined by
equilibrium binding and kinetics are virtually identical provides
assurance of the accuracy of these values. Thermodynamic parameters
determined from these K
measurements
display, for all but one FA, predominant -T
S values, which increase monotonically from -6.4 to -3.7
kcal/mol with increasing double bond number from oleate to
arachidonate, whereas the enthalpic contributions are all between about
-3 and -4 kcal/mol (). The absolute values as
well as the relative entropic and enthalpic contributions are
significantly different from the values obtained for the unlabeled
FABPs, for which the enthalpic and entropic contributions are between
about -12 and -5 kcal/mol and -4 and +3,
respectively (I). In particular, while the ADIFAB
enthalpic contribution is between about 7 and 9 kcal/mol more positive
than its parent protein I-FABP, its entropic contribution is between
about 6 and 7 kcal/mol more negative ().
changes upon FA
binding in a manner that might force the attached acrylodan from a
highly restricted position within the portal region to one that is
mobile and almost orthogonal to the protein's surface (data not
shown). The much smaller ADIFAB net enthalpy (average
H
= 7 kcal/mol)
probably results because the strong interactions between the acrylodan
and protein, which are required to immobilize the acrylodan, must be
disrupted before the FA can enter the cavity. This would suggest, if
derivatizing Lys
with acrylodan does not affect the
conformation of the cavity, that acrylodan should primarily reduce the
on rate of FA binding to apoADIFAB but have little effect on the off
rate. Preliminary studies of the binding kinetics are, in fact,
consistent with this prediction (Ref. 12 and data not shown).
Natural FABPs
FABPs from adipocyte, intestine, and
heart exhibit similar binding thermodynamics (I). In
almost all instances, the free energy is dominated by an enthalpic
term. Previous calorimetry studies also found substantial enthalpic
contributions for FA binding to FABP. Oleate binding to I-FABP was
found to have a H of -8.1 and a
-T
S of +1 kcal/mol(37) , and
oleate and arachidonate binding to A-FABP were found to have
H values of -7.8 and -7.4 kcal/mol and
-T
S values of +1.7, and +0.6
kcal/mol, respectively(14) . Although the present study,
indicating large enthalpic and small entropic changes (I),
is in qualitative agreement with these calorimetry results,
H values in the present study are more negative by about 2-4
kcal. The weaker interaction observed by calorimetry may be a
consequence of either the presence of organic solvent added with the FA
or the use of FA concentrations that greatly exceed their solubility
limits(12) .
values,
G values follow
the pattern:
G (adipocyte) >
G (intestine) >
G (heart). For a given FABP,
G generally increases with the aqueous solubility of the
FA; for the same FA chain length,
G increases
monotonically with double bond number (I). The results in I also indicate a trend in which, for each FABP,
-T
S increases with increasing FA double
bond number and
H decreases with increasing double bond
number. For example in the case of H-FABP, -T
S increases by about 6 kcal/mol and
H decreases by 5
kcal/mol from oleate to arachidonate. These results suggest a
favorable, perhaps additive, enthalpic contribution involving FA double
bonds that is offset by the unfavorable entropic contribution,
consistent with increasing aqueous solubility with double bond number.
Overall Thermodynamics of Binding
Although binding
of different FA and FABPs reveal substantial differences in affinities,
this heterogeneity involves relatively small differences in energy
(3 kcal/mol), as is apparent from the variation of the
thermodynamic parameters shown in I. Elucidation of the
specific mechanisms responsible for this heterogeneity will require
measurements involving site-specific mutants and the development of
more accurate theoretical methods for relating energetics and
structure. At this stage, however, a rough analysis can be applied to
identify the dominant interactions that govern FA binding to FABP.
G
) as a sum of free
energies corresponding to the individual interactions that contribute
to
G
, as shown by Equation
9.
G
represents the free energy
difference between bulk solvent and bulk solvent + FA. The free
energy difference between the bound FA-FABP state and the one in which
both FA and FABP are free is separated into four terms:
G
is the difference between FA bound
to FABP and in bulk solvent,
G
is the
difference between holoFABP and apoFABP,
G
is the interaction energy between the FA and FABP, and
G
is the energy difference between
specific free water molecules and these molecules bound to FABP. We now
consider each of these contributions in turn.
Bulk Solvent Free Energy (
G
)
Free Energy of Transfer of FA between Water and
Membrane (
The free energy
change of the bulk solvent (G
)
G
) that appears
in Equation 9 is identical to the change in bulk solvent that occurs in
the transfer of FA from water to membrane. In what follows,
G
will be estimated from the free energy of
FA partition into membranes. The free energy difference for this latter
process (
G
) is composed of
contributions from: 1) bulk solvent (
G
), 2)
the difference between FA in the membrane and in bulk solvent
(
G
), and 3) the difference in the
membrane free energy with and without FA bound
(
G
). Thus, we obtain Equation
10.
G
was determined from the
measured partition coefficients as described under ``Experimental
Procedures.'' The
G
values () range from a high of -8.6 kcal/mol for linolenate,
the most soluble FA, to -10.1 kcal/mol for palmitate, the least
soluble of the FA. These values are in good agreement with the
-9.8 kcal/mol value obtained for palmitate transfer into lipid
vesicles by Peitzsch and McLaughlin (27) and with the -9
kcal/mol value for palmitate determined for transfer into n-heptane by Tanford (38). The lack of temperature dependence
of partition () and, therefore, a partition that is
primarily entropy driven is also in agreement with the results of
Peitzsch and McLaughlin(27) .
Free Energy Change of the Membrane
(
Several observations suggest
that the free energy change in the membrane
(G
)
G
) due to FA insertion is small. The
lipid order of the egg phosphatidylcholine-composed large unilamellar
vesicle bilayers is virtually unaffected by the insertion of FA, at
least at low FA concentrations(21) , suggesting that
G
0. Moreover, studies of the binding
of transmembrane peptides, which are probably much more perturbing to
membranes than are FA, are also consistent with a small
G
(about 2 kcal/mol) because of
compensating entropic and enthalpic peptide-lipid
interactions(39) . Therefore, to a reasonable approximation,
G
G
+
G
.
Free Energy Change of the FA
(
Two terms are
expected to contribute to G
)
G
: 1) the
change in the rotational and translational motion of the FA molecule as
a whole due to immobilization by the membrane and 2) the change in FA
intramolecular interactions in the membrane as compared to the water.
Previous studies have suggested that considerable translational and
rotational immobilization might occur when a molecule is transferred
from water to membranes, and that this reduction in mobility would be
associated with an appreciable (>10 kcal/mol for FA) increase in
free energy(39, 40) . These estimates appear, however,
to be greater than the actual free energy cost of this
transfer(41) . This is supported by the results of Peitzsch and
McLaughlin(27) , who compared the membrane partition of the
uncharged FA with transfer of FA between water and n-heptane (38) and concluded that the free energy cost of FA
immobilization by the membrane was negligible. The second potential
contribution to
G
, due to the change in
the internal energy of the FA upon transfer into the membrane, is also
likely to be small because the interactions between FA and water and FA
and lipid are relatively weak. This speculation is supported by the
negligible overall enthalpy change upon transfer between water and
membrane. Because
H
is also
0 at 25
°C(38) ,
H
0
presumably reflects the small magnitude of the FA-membrane
interactions, although exactly compensating changes can not be ruled
out. We conclude, nevertheless, that both potential contributions are
negligible and therefore
G
0
kcal/mol.
(
Because all terms
except G
)
G
in equation (10) are assumed
to be negligible,
G
G
. As seen in ,
G
and therefore
G
values ranges from -8.6 to
-10.1 kcal/mol. Thus a significant free energy gain is obtained
by pushing these long chained FA out of the water and into membranes.
The same contribution of water (
G
) for
membrane transfer must also apply to the transfer of FA from water to
the FABP binding sites.
Thermodynamics of FA Binding to FABP
Free Energy Change of the FA
(
Having evaluated
G
)
G
(), we now consider the
remaining terms in Equation 9. The contribution
G
is analogous to the term
G
for membranes, and here again this term
is composed of two contributions: the first due to the change in the
motion of the FA as a whole and the second due to the change in the
conformational energy of the FA. As discussed above, the free energy
contributions due to immobilization of molecules upon transfer from
water to membranes have been overestimated in previous studies. The
effect of such contributions for ligand binding to proteins has been
investigated recently by Murphy et al.(42) . Applying
experimental and theoretical arguments to the analysis of three
separate peptide systems, Murphy et al. concluded that
-T
S for the translational component of the
free energy change, as would apply for FA binding to FABP, is +2.4
kcal/mol. Estimating the contribution to
G
that is due to reduction in the rotational mobility according to
Finkelstein and Janin (41) and modeling the FA as a rod-like
rotor, we obtain a value for this contribution of about +1
kcal/mol. Thus we estimate that the free energy change due to the
decrease in the mobility of the FA as a whole upon binding to FABP is
about +3 kcal/mol.
G
, the change in internal free energy
of the FA, is a sum of changes in configurational enthalpy and entropy.
Determining these quantities requires information about the average
conformation and dynamics of the FA in water and bound within the FABP.
While the crystal coordinates and temperature factors of a number of FA
bound within FABPs are known, similar information is not available for
the FA in water. Recently, Rich (43) has used molecular dynamics
to explore FA configurations in vacuum and these results indicate that
the maximum energy differences between different FA conformations in
vacuum are about +13 kcal/mol. Using the crystal structures of
stearate and oleate in adipocyte FABP, Rich
(
)has
estimated that the enthalpy differences between the minimum energy
configurations of these FA in vacuum and their FABP conformations are
about +5 kcal/mol. Although similar estimates of the entropy
changes are not available, it is likely that rotations about single
carbon bonds are more restricted in the protein than in the water.
Assuming complete rotational freedom about single C-C bonds in water
but none in the protein, -T
S would range
between about +12 kcal/mol for stearate (18:0) to 8 kcal/mol for
linolenate (18:3), using an entropy contribution of Rln3 for each
carbon(41) . Certainly without information about the aqueous
conformational freedom of the FA and possible contributions from
vibrational degrees of freedom, there is considerable uncertainty in
these estimates. For the discussion below, however, we assume a
configurational enthalpy change of +5 kcal/mol, a configurational
entropy change of +10 kcal/mol, and a +3 kcal/mol entropy
loss due to the decrease in the overall FA mobility. Together, these
contributions yield a total
G
of
+18 kcal/mol.
The Free Energy Change of the FABP
(
The term
G
)
G
represents that portion of the free
energy difference between the holoFABP and apoFABP that is limited to
structural differences of the polypeptides, contributions due to
interactions with FA and water are treated separately. Crystallographic
studies reveal that the apoFABP structure is virtually unchanged by FA
binding; root-mean-square main chain and side chain positions in the
apo and holo proteins are generally less than about 0.5Å and
1Å, respectively(7, 14) . Although a few residues
are discretely disordered in the apo as compared to the holo
structure(6, 7) , this change is unlikely to contribute
significantly to the free energy difference because the apo structure
itself is highly ordered. Thus, because FABP structure changes only
slightly upon FA binding,
G
is probably
small and we assume for this discussion that
G
0.
FA-FABP Interactions
(
The term
G
)
G
represents interactions between
specific amino acid side chains, possibly in complex with bound water
molecules, and specific portions of the FA. One of the most clearly
defined interaction involves the FA carboxylate and amino acid side
chains, most importantly Arg
and Arg
, and
ordered water molecules(6, 7) . Mutations of the
residues involved in this network reduce the binding affinities by
about 20-50-fold, suggesting that this interaction contributes
between -1.8 and -2.3 kcal/mol to
G
(37, 44) . In addition
to this interaction, which is localized to the carboxylate portion of
the FA, the variation of binding affinities with FA double bond number
suggests that an interaction between the hydrocarbon chain of the FA
and the hydrophobic residues within the cavity also contributes to
G
(12) . Crystallography results
also suggest specific interactions between the terminal methyl portion
of the FA and residues in the proposed portal region of the protein, as
well as specific interactions between bound water molecules and the
fatty acid's aliphatic chain(6, 7, 14) .
Estimates of enthalpic or entropic contributions from these and other
interactions that probably play important roles in binding are not yet
available. Thus, for the
G
contribution, we assume a -2 kcal/mol enthalpic
contribution due to the carboxylate interaction, because binding to the
Arg mutants is reduced about 50-fold, plus unknown enthalpic (HI) and
entropic (SI) contributions due to interactions between the FA and FABP
at other regions along the hydrocarbon chain.
Bound Water Interactions
(
The term
G
)
G
represents the energy change due to
the displacement of ordered water molecules by the FA. Two
contributions to
G
can be identified:
the increase in entropy resulting from the transfer to bulk solvent and
the enthalpic cost of breaking hydrogen bonds with amino acid side
chains and waters remaining bound within the cavity. Reasonably well
defined limits have been suggested for entropy changes due to water
binding to proteins. Previous studies have found that hydration
entropies for inorganic salts range between 0 and 2 kcal/mol, the
largest values corresponding to the most ordered molecules with
temperature factors of 5-10 Å
, and because they
are independent of the salt composition, these values have been
suggested to apply equally well to proteins(45) . Water
molecules bound to FABP, however, have significantly larger temperature
factors than found in salt crystals; most are greater than 20
Å
(46) . Using these larger temperature factors
and following the approach of Finkelstein and Janin(41) , the
estimated entropy change per bound water molecule is small (
0). The
range of enthalpic contributions is more difficult to estimate because
of the wide range of hydrogen bonding energies between water and
charged or polar side chains(47) . Moreover, the breaking of
hydrogen bonds between water and amino acid side chains may be
compensated by the reformation of hydrogen bonds in the bulk solvent.
Thus displacement of water from the FABP cavity by FA may contribute
relatively little to the free energy of binding, and we assume for this
discussion that
G
0.
Comparison of the Measured Thermodynamic Parameters
with the Sum of Terms in Equation 9
G
= -10,
H
= -10, and
-T
S
= 0
kcal/mol. These measured values, together with the estimates of the
terms of Equation 9, discussed above, are listed in . The
entries in this table explicitly identify those estimates for which we
believe finite but as yet unknown contributions (SI and HI) may be
significant. As the results in suggest, only two terms
are likely to contribute significantly to the large net favorable
enthalpy of binding (-10 kcal/mol). One of these
(
G
) is a substantial unfavorable
(positive) contribution resulting from the energy difference of the FA
configuration in vacuum and bound within the protein.
The
second contribution is the enthalpy of FA-FABP interaction, which in
addition to the -2 kcal/mol carboxylate-FABP contribution
includes a term HI, representing all the other interactions between the
FA and protein. It follows from the entries in that HI is
on the order of -13 kcal/mol and, therefore, that binding to FABP
involves FA-FABP interactions with magnitudes greatly exceeding those
involving the FA carboxylate head group. Summation of the entropic
contributions listed in suggests that the small net
entropy of binding results from the sum of the large favorable entropy
change caused by the hydrophobic effect (-10 kcal/mol) and
substantial reductions in the fatty acid's conformational entropy
as well as possible reductions in amino acid side chain configurational
entropy as a consequence of interactions with the FA (SI).
G
. The term
G
itself, based on the difference
between FA bound to FABP and in the vacuum state rather than in water,
may not be accurate. On the other hand, although the magnitude of this
term may diminish with improved calculation, it is unlikely that a
negative contribution would be obtained; some substantial negative
enthalpic interaction is required to account for the measured -10
kcal/mol value. Thus, although the magnitude of the results that follow
from may be altered by improved estimates of the
individual interactions, unless other sources of favorable enthalpy and
unfavorable entropy can be identified, the HI and SI terms likely
contribute significantly to the thermodynamics of binding.
-10 kcal/mol) enthalpic term that
dominates the overall free energy of binding
(
G
) also results from the
competition of at least two substantial terms: the unfavorable FA
configurational enthalpy (
+5 kcal/mol) and the favorable
enthalpy of the FA-FABP interaction (2 kcal/mol + HI). At present,
only one specific interaction can be identified for which an enthalpy
value can be assigned: the carboxylate-hydrogen bonding network,
which probably contributes about - 2 kcal/mol to the total
approximately -13 kcal/mol expected
H value. The
comparison of palmitate and oleate binding discussed above suggests
that the additional enthalpic interactions involve the hydrocarbon
portion of the FA chain and the amino acid side chains and bound water
molecules that interact with this part of the FA, a conclusion also
reached by LaLonde et al.(14) in their study of the
adipocyte FABP. Identifying the specific interactions that account for
the remaining approximately -11 kcal/mol will require
measurements of the binding energies of a variety of ligands with
site-specific FABP mutants coupled with corresponding structural
information. Nevertheless, the analysis even at this early stage is
significant because it predicts that changes in the amino acid side
chains in contact with the hydrocarbon portion of the FA chain should
lead to very large changes in the enthalpy of binding.
Table: ADIFAB
thermodynamic parameters of FA binding
G and -T
S were
determined at 25 °C.
Table: 1652105331p4in
Dissociation constants determined from the
fit to data in Fig. 1.(119)
Table: Thermodynamic parameters of FA binding
Table: I-FABP-ADIFAB differences
Table: Free energies associated with FA
partition into membranes
Table: Comparison of measured and estimated
thermodynamic parameters of FA binding
G,
H, and -T
S shown in this
table were estimated as described in the text. The entries in Total are
the sums of the preceding five terms in each of the three columns. Each
of the terms represent the following changes upon FA binding: bulk
solvent (Water), overall and internal conformations of the FA (FA),
changes in the FABP conformation (FABP), FA-FABP interactions
(FA-FABP), and water bound within the FABP cavity (BW-FABP). The terms
HI and SI represent the as yet unidentified FA-FABP interactions, whose
enthalpy (HI) and entropy (SI) contributions are required to balance
the measured = total equation; thus HI = -13 and SI
= -3 kcal/mol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.