From the Department of Physiology and Pharmacology, Texas A & M University, TVMC, College Station, Texas 77843-4466
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ABSTRACT |
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Cellular unbound long chain fatty acyl-CoAs (>14
carbon) are potent regulators of gene transcription and intracellular
signaling. Although the cytosolic acyl-CoA binding protein (ACBP) has
high affinity for medium chain fatty acyl-CoAs, direct interaction of
ACBP with >14-carbon fatty acyl-CoAs has not been established. Steady
state, photon counting fluorescence spectroscopy directly established
that rat liver ACBP bound 18-carbon cis- and
trans-parinaroyl-CoA, Kd = 7.03 ± 0.95 and 4.40 ± 0.43 nM. Time-resolved fluorometry revealed that ACBP-bound parinaroyl-CoAs had high rotational freedom within the single, relatively hydrophobic ( <32), binding site. Tyr
and Trp fluorescence dynamics demonstrated that apo-ACBP was an
ellipsoidal protein (axes of 15 and 9 Å) whose conformation was
altered by oleoyl-CoA in the holo-ACBP as shown by a 2-Å decrease of
ACBP hydrodynamic diameter and increased Trp segmental motions. Thus,
native liver ACBP binds >14-carbon fatty acyl-CoAs with nanomolar
affinity at a single binding site. Acyl-CoA-induced conformational
alterations in ACBP may be significant to its putative functions in
lipid metabolism and regulation of processes sensitive to unbound long
chain fatty acyl-CoAs.
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INTRODUCTION |
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It is now recognized that long chain fatty acyl-CoAs (>14 carbons) serve at least four essential cellular functions as follows: fatty acid oxidation (1-7), fatty acid esterification (8-16), signal transduction (17-19), and gene expression (20, 21). Although cellular long chain fatty acyl levels are normally in the range 5-164 µM, these levels increase as much as 4-fold in pathological conditions (reviewed in Ref. 22). Since the critical micellar concentration of long chain fatty acyl-CoAs is 30-60 µM and as much as 96-99% of long chain fatty acyl-CoA partitions to membranes, this suggests that high levels of long chain fatty acyl-CoAs may disrupt membrane function as well as cellular signaling/gene regulation (reviewed in Refs. 10 and 22).
Because of these considerations, it is important to understand the mechanisms that regulate the distribution of intracellular free long chain fatty acyl-CoAs. One candidate cytosolic protein that may serve this purpose is ACBP1 which may sequester, store, and/or protect fatty acyl-CoAs from hydrolysis (reviewed in Ref. 22). Acyl-CoA binding protein (ACBP) is a small (10 kDa), highly conserved cytosolic protein broadly distributed among all eukaryotic tissues studied, and elevated expression of ACBP has been observed in malignant tumors and transformed cells (reviewed in Refs. 22-24).
A primary objective of the present investigation was to use a fluorescent fatty acyl-CoA binding assay as well as non-fluorescent oleoyl/acyl-CoA to directly show for the first time that native rat liver ACBP actually bound the most common chain length, C18, fatty acyl-CoAs. The second aim was to examine the structural dynamics of native rat liver apo- and holo-ACBP, to characterize the properties of fluorescent fatty acyl-CoAs within the ACBP-binding site, and to examine ACBP conformational dynamics in response to ligand binding.
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EXPERIMENTAL PROCEDURES |
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Materials
cis- and trans-parinaroyl-CoAs were synthesized as described (9). cis- and trans-parinaric acids were from Calbiochem. Coenzyme A (CoASH), acyl-coenzyme A synthase, ATP, p-terphenyl, and 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene, and saturated and unsaturated fatty acyl-CoAs were from Sigma. All other chemicals were reagent grade or better.
Methods
ACBP was isolated from rat liver as described previously (24). Protein concentration was determined by the Bradford assay (25) and corrected according to UV spectrophotometric analysis (26). The Bradford assay overestimates ACBP concentration by 1.69-fold.
Steady State Fluorescence Spectroscopy
Steady state fluorescence spectra were measured in a 1-cm quartz
cuvette with a PC1 Photon Counting Spectrofluorimeter (ISS Instr.,
Champaign, IL). Sample temperature was maintained at 25 °C (±0.1 °C). Excitation and emission bandwidths were 4 and 8 nm. Sample absorbance at the excitation wavelengths was 0.05.
Time-resolved Fluorescence Spectroscopy
Data Acquisition--
All measurements of lifetime, differential
polarized phase (limiting anisotropy, rotational rate), hydrodynamic
radius, and wobbling in a cone angle were performed as described
earlier with a GREG 250 Subnanosecond Multifrequency Cross-correlation
and Modulation fluorometer with KOALA automatic sample compartment (ISS
Instruments, Champaign, IL) (27-29). Intrinsic ACBP aromatic amino
acids were excited with an Innova-Sabre argon ion laser (Coherent Laser
Group, Palo Alto, CA) with automatic wavelength selection at = 275.4, 300.2, 302.4, and 305.5 nm (power output 340, 630, 800 and 460 milliwatts, respectively). ACBP intrinsic fluorescence was observed
through 313 BP10 and 341 BP15 interference filters (Omega Optical Inc.,
Brattleboro, VT). The extrinsic fluorescent ligands, cis-
and trans-parinaroyl-CoAs, were excited at 325 nm by a 424NB
He-Cd laser (Liconix Inc., Sunnyvale, CA), and emission was observed
through a KV389 low fluorescent cut-off filter (Schott Glass
Technologies, Duryea, PA). Sample absorbance at the excitation wavelengths was
0.05. All data were obtained in 25 mM
phosphate buffer, pH 7.4, at 25 °C.
Lifetime Data Analysis-- Lifetime data were analyzed by ISS-187 Software (ISS Inc., Champaign, IL) as a sum of exponentials shown in Equation 1.
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(Eq. 1) |
Anisotropy Decay Data Analysis--
Anisotropy decay was modeled
by a sum of exponentials as follows: r(t) = r(0)gi
exp(
t/
i), where r(0) is anisotropy
of a fluorophore in the absence of rotational diffusion,
i
is the rotational correlation time, and gi is
fractional anisotropy. The "goodness" of fit to the applied model
was determined as described above using ISS-187 Software (ISS
Instruments, Champaign, IL). The equivalent hydrodynamic radius of the
protein was calculated as shown in Equation 2.
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(Eq. 2) |
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(Eq. 3) |
Fluorescent Fatty Acyl-CoA Binding Assay
ACBP binding affinities for parinaroyl-CoAs were determined as described previously (28, 32) with the following modifications. A 2-ml sample of 0.05 µM ACBP in phosphate buffer was titrated with small increments of fatty acyl-CoA (0.1-1.0 µl) dissolved in double-distilled water. The fatty acyl-CoA stock solution concentrations were 270-300 µM. Each sample and blank (without ACBP) were thoroughly mixed and allowed to equilibrate for 1-2 min to permit stable measurement of the fluorescence signal. All measurements were performed at 25 °C.
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RESULTS |
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Fluorescent Fatty Acyl-CoA Binding to Native Rat Liver ACBP
The binding affinity of native rat liver ACBP for fatty acyl-CoA was examined using a direct binding assay. This assay takes advantage of CoA derivatives of naturally occurring fluorescent parinaric acids and does not require separation of bound from free fatty acyl-CoA. As shown in Fig. 1, cis- and trans-parinaroyl-CoA are both 18-carbon fatty acids with 4 conjugated double bonds near the methyl terminus of the fatty acyl chain. cis-Parinaroyl-CoA has a cis-double bond at position 9, the same as that of the 18-carbon oleoyl-CoA (Fig. 1). Consequently, the kinked shape of the cis-parinaroyl-CoA acyl chain is nearly superimposable on that of the oleoyl-CoA acyl chain. In contrast, trans-parinaroyl-CoA has a straight chain fatty acyl chain, just like that of the 18-carbon stearoyl-CoA acyl chain (Fig. 1). Consequently, the straight shape of the trans-parinaroyl-CoA acyl chain is nearly superimposable on that of the stearoyl-CoA acyl chain. These properties make cis- and trans-parinaroyl-CoA excellent fluorescent structural analogues oleoyl- and stearoyl-CoA, respectively.
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Although cis- and trans-parinaroyl derivatives fluoresce poorly in aqueous solution, upon titration of ACBP with cis- or trans-parinaroyl-CoA the fluorescence intensity increased, consistent with the ligand becoming localized in a more hydrophobic environment, i.e. protein binding pocket (Fig. 2). No shifts of cis- or trans-parinaroyl-CoA fluorescence emission maxima were detected upon titration of ACBP (data not shown). Therefore, the increase in fluorescence emission intensity of the parinaroyl-CoAs, measured at 420 nm after each addition of ligand to ACBP, was corrected for the background (no protein) and plotted as a function of total ligand concentration. This procedure yielded pure saturation curves for both cis- and trans-parinaroyl-CoA as shown in Fig. 2, A and B, respectively. Maximal fluorescence intensities were determined by titrating a small amount of each parinaroyl-CoA with increasing ACBP. These data were then used to construct Scatchard plots (Fig. 2, A and B, insets) and obtain binding parameters, dissociation constant (Kd), and molar stoichiometry (Bmax). Native rat liver ACBP bound cis- and trans-parinaroyl-CoAs at a single binding site: cis-parinaroyl-CoA Bmax = 0.93 ± 0.04 mol/mol, trans-parinaroyl-CoA Bmax = 0.89 ± 0.01 mol/mol. The respective affinities were Kd = 7.03 ± 0.95 and 4.4 ± 0.43 nM. The binding affinity of ACBP for the straight chain trans-parinaroyl-CoA was significantly 1.5-fold higher than that for the kinked chain cis-parinaroyl-CoA. In summary, these data provide the first direct evidence that ACBP binds naturally occurring 18-carbon chain fatty acyl-CoAs with nanomolar affinity.
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Steady State Fluorescence Properties of Native Rat Liver Apo-ACBP
Rat liver ACBP is a protein with multiple intrinsic fluorophores,
four Tyr and two Trp amino acid residues (24), which may result in a
heterogeneous fluorescence emission of ACBP. Indeed, upon excitation at
270 nm, the wavelength where both Tyr and Trp were excited, native rat
liver apo-ACBP had a rather broad (bandwidth 65 nm) fluorescence
emission spectrum with a maximum at 322 nm and a shoulder near 300 nm
(Fig. 3). Shifting the excitation
wavelength to 280 nm resulted in a 7-nm (bandwidth 72 nm) broadening of
the emission spectrum, almost exclusively on its blue edge. However, there was no effect on the position of the emission maximum
(max = 322 nm) (Fig. 3). Further shifting the excitation
wavelength to 296 nm, the wavelength where Trp residues were
preferentially selectively excited, the apo-ACBP emission maximum
(
max = 332 nm) underwent a dramatic (~10 nm)
bathochromic shift, and the emission spectrum was narrowed by almost 11 nm (bandwidth 61 nm), as compared with that of excitation at 280 nm
(Fig. 3).
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In summary, the fluorescence emission characteristics of apo-ACBP
proved to be strongly dependent on excitation wavelength. The data
showed that apo-ACBP fluorescence spectra had a distinct Tyr emission
component as evidenced by an unusually short wavelength emission maxima
(max = 322 nm) upon excitation at 270 and 280 nm, as
well as by the appearance of a characteristic Tyr emission shoulder
near 300 nm upon excitation at 270 nm. Concomitantly, the apo-ACBP Trp
emission component was clearly identified by its emission maximum at
332 nm upon excitation at 296 nm. The fact that native rat liver
apo-ACBP had rather broad emission spectra (bandwidth 60-70 nm),
regardless of the excitation wavelength (270-296 nm), was also
consistent with both Tyr(s) and Trp(s) being present in highly
heterogenous loci within the ACBP protein. In the case of the apo-ACBP
Trp residues, these loci were characterized as highly hydrophobic, as
shown by the fluorescence emission maximum of 332 nm (excitation at 296 nm) (33).
Effect of Oleoyl-CoA Binding on Steady State Fluorescence Characteristics of Native Rat Liver ACBP
The effect of oleoyl-CoA binding on fluorescence emission
characteristics of native rat liver ACBP aromatic amino acid residues was monitored by exciting the protein emission at two wavelengths, 270 and 296 nm. The excitation at 270 nm resulted in emission from both Tyr
and Trp components, whereas the 296-nm excitation allowed observation
primarily of ACBP Trp emission (see above). As shown in Fig.
4A, upon excitation at 270 nm,
the fluorescence emission of ACBP was progressively quenched (up to
2-fold) upon titration apo-ACBP with increasing amounts of
non-fluorescent oleoyl-CoA to form holo-ACBP. The reduction in
fluorescence intensity was accompanied by a gradual red shift of the
ACBP emission maximum (Fig. 4A). The latter shifted almost 8 nm (from 322 to 330 nm), when oleoyl-CoA was present in 10-fold molar
excess over ACBP (Fig. 4A). The disappearance of both the
shoulder observed near 300 nm in the ACBP emission spectrum (Fig.
4A) and the gradual spectral shift of the emission maximum
to the position of the Trp emission maximum (max = 330 nm, see Figs. 3 and 4A) were consistent with oleoyl-CoA
predominantly quenching the Tyr fluorescence emission component of
ACBP. This suggestion was further supported by results of experiments
where the same titration was repeated with excitation at 296 nm where
Trp, rather than Tyr, was selectively excited (Fig. 4B). In
contrast to titration with oleoyl-CoA and 270-nm excitation (Fig.
4A), ACBP fluorescence emission and the position of its
maximum (
max = 332 nm) were not significantly changed by
oleoyl-CoA binding (Fig. 4B) when the ACBP was excited at
296 nm. Thus, ACBP Trp residues did not appear available for quenching
induced by the bound oleoyl-CoA. In summary, the differential effects
of oleoyl-CoA on ACBP Tyr and Trp fluorescence emission characteristics
were consistent with the following: (i) ACBP Tyr, but not Trp,
residue(s) being localized within the oleoyl-CoA binding pocket, and/or
(ii) ligand-induced conformational changes in the ACBP tertiary
structure. These possibilities were resolved in the following
sections.
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Time-resolved Fluorescence
Fluorescence Lifetime of Apo- and Holo-ACBP--
Time-resolved
fluorescence studies of apo- and holo-ACBP were performed to understand
better the nature of ACBP emission as well as the processes occurring
in the protein globule on the pico- to nanosecond time scale upon
ligand binding. The fluorescence decay of apo-ACBP was biexponential in
nature, consistent with the presence of several emitting centers in the
protein. Upon exciting at 275 nm (both Tyr and Trp excitation) but
measuring emission at 313 nm (i.e. preferential measurement
of ACBP Tyr emission at the blue edge of ACBP emission spectrum, Fig.
3), the major lifetime component was 1 = 0.76 ns
(fraction 0.82), and the minor component was
2 = 2.44 ns
(Table I). This biexponential decay
resulted in a mean lifetime of <
> = 1.06 ns (Table I). Addition of
10 molar eq of oleoyl-CoA to form the holo-ACBP reduced both the long
lifetime value, from 2.44 to 0.79 ns, and even more dramatically the
short component from 0.76 to 0.18 ns, while increasing the fraction of
the long lifetime component by almost 4-fold, from 0.18 to 0.68. The
overall effect of oleoyl-CoA binding to ACBP was a reduction of the
average lifetime by approximately 2-fold, from 1.06 to 0.59 ns (Table
I).
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Anisotropy of Apo- and Holo ACBP--
Time-resolved fluorescence
anisotropy was used to examine the rotational dynamics of native rat
liver apo-ACBP Tyr. Upon excitation at 275 nm and measurement of
fluorescence emission at 313 nm, ACBP exhibited monoexponential
anisotropy decay kinetics with a rotational correlation time = 3.07 ns (Table II). This relatively long
correlation time reflected overall protein motion and was consistent
with that expected for protein (ACBP) with molecular mass near 10 kDa
(31). The ACBP hydrodynamic radius calculated from the time-resolved
anisotropy data was 15.1 Å (Table II). The fact that the measured
residual (limiting) anisotropy of ACBP r = 0.156 (Table
II) was much less than both the maximal Trp and Tyr anisotropies
measured in a propylene glycol glass at
60 °C, r0 = 0.315 (34), and r0 = 0.320 (35), respectively, indicates that ACBP aromatic amino acid local
segmental motions were relatively fast and not resolvable on the
nanosecond time scale. In addition to this fast rotation, the estimated
amplitude for such rotational motions, i.e. the
"wobbling" in cone angle, was also large, near 36° (Table
II).
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Time-resolved Fluorescence of cis- and trans-Parinaroyl-CoAs in the ACBP Fatty Acyl-CoA-binding Site-- The molecular dynamics of the fatty acyl-CoA ligand within the ACBP-binding site were examined. cis- and trans-parinaroyl-CoA were bound to ACBP, and their fluorescence intensity and anisotropy decays were determined.
Fluorescence Lifetime--
As shown in Table
III, ACBP bound cis- and
trans-parinaroyl-CoAs both displayed complex fluorescence
decay kinetics which were best fit (2 <3) to three
exponentials. The absolute values of the longest fluorescence decay
components
1 ~70 ns presented in Table III must be
considered as their lowest limit. The best fits (
2 <3)
of the respective emission kinetics yielded a range of values, 70-500
ns, for this slowest component of the different emission decay curves,
without changing the other components and their respective fractions
(data not shown). The calculated average lifetimes for cis-
and trans-parinaroyl-CoA bound to ACBP were 10.55 and 7.38 ns, respectively (Table III). These average lifetime values were
significantly longer than that of cis-parinaroyl-CoA in
ethanol, <
> = 4.5 ns (27). These observations were consistent with
the fluorophore (tetraene located between C10 to
C18 of the fatty acyl chain) of cis- and
trans-parinaroyl-CoA being rather shielded from the aqueous
solution when bound to ACBP. Furthermore, the longer average lifetime
of cis-parinaroyl-CoA bound to ACBP also suggested that the
cis-parinaroyl-CoA was localized in a more hydrophobic
and/or restricted microenvironment as compared with the
trans-parinaroyl-CoA (shorter average lifetime).
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Fluorescence Anisotropy--
The analysis of anisotropy decay of
cis- and trans-parinaroyl-CoAs bound to ACBP
revealed single exponential kinetics for both ligands. Both molecules
exhibited similar rotational correlation times, = 3.30 and 3.37 ns,
respectively, within the ACBP fatty acyl-CoA-binding site (Table
IV). It should be noted that the rotational correlation time values observed for parinaroyl-CoAs bound
to ACBP were nearly identical to those obtained for ACBP protein
overall rotations, based on the intrinsic ACBP fluorescence upon
excitation at 275 nm (Table II). Therefore, the rotational correlation
times measured for the bound parinaroyl-CoAs (Table IV) can be assigned
to the rotational motions of the holo-ACBP. The respective hydrodynamic
radii for ACBP bearing cis- or
trans-parinaroyl-CoAs were calculated to be ~15.5 and 15.6 Å (Table IV).
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DISCUSSION |
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Although the amino acid sequence of ACBP is relatively conserved among a variety of species, to date much of our knowledge of ACBP is limited to the bovine. Data from other fatty acyl-CoA binding protein families indicate that an alteration of even a single amino acid residue may dramatically alter its ligand specificity (37-39) and/or structure/function (reviewed in Refs. 27 and 39-41).
The present study used a direct binding assay to demonstrate for the
first time that native rat ACBP binds the naturally most prevalent
chain length (18 carbon) fatty acyl-CoAs with nanomolar affinity. This
finding is especially significant since examination of fatty acyl-CoA
binding affinities over the past several years focused largely on
16-carbon fatty acyl-CoAs binding to bovine ACBP. Moreover, the
actual affinity of bovine ACBP for the most common naturally occurring
long chain (>14 carbon) fatty acyl-CoAs is uncertain (22). Part of the
problem lies in the lack of consistency of different fatty acyl-CoA
binding assays which have yielded Kd values ranging
from 10
6 to 10
14 M.
Furthermore, even state of the art binding assays such as microcalorimetry could not provide a direct binding assay for >14-carbon fatty acyl-CoAs, whereas an indirect (displacement) microcalorimetry binding assay required a number of assumptions to
yield reasonable data for bovine ACBP and the 16-carbon
hexadecanoyl-CoA, ranging nearly 5 orders of magnitude (22, 42). As
shown herein, native rat liver ACBP had high affinity, nM
Kd, for the naturally occurring C18
kinked (cis-parinaroyl-CoA) and C18 straight (trans-parinaroyl-CoA) acyl-CoAs: 7.03 and 4.4 nM, respectively. Consistent with this, an indirect
microcalorimetric binding assay concluded that bovine ACBP bound
C16 palmitoyl-CoA with a Kd = 2 nM (42) while another indirect binding assay (membrane
partitioning/competition) yielded a Kd = 5 nM for bovine ACBP (10, 42). Additionally, analysis of
cis-parinaroyl-CoA displacement data by a series of saturated and unsaturated acyl-CoAs with different chain lengths revealed that rat liver ACBP binds saturated fatty acyl-CoAs with chain
lengths ranging from C12 to C20 and unsaturated
fatty acyl-CoAs with chain length from C14 to
C20.2
The same fluorescent method and ligands were used to assess the binding affinities of ACBP, sterol carrier protein-2 (29) and liver fatty acid binding protein (39). This allows direct comparison of ACBP, SCP-2, and L-FABP long chain fatty acyl-CoA binding affinities. The binding Kd values of SCP-2 for cis- and trans-parinaroyl-CoA were reported to be 4.57 and 2.76 nM (29). The same parameters measured for the L-FABP high affinity binding site yielded 8 and 10 nM Kd values, as well as 97 and 180 nM Kd values for the L-FABP low affinity binding site (39). Thus, it appears that among these proteins SCP-2 had a slightly higher binding affinity for parinaroyl-CoAs, followed by ACBP and then by L-FABP. Interestingly, both ACBP and SCP-2 (but not L-FABP) exhibited a similar trend; they bound straight chain trans-parinaroyl-CoA tighter than the kinked chain counterpart. In summary, all three fatty acyl-CoA proteins would be expected to play a role in determining cellular fatty acyl-CoA partitioning between membranes and aqueous/cytosolic compartments, although the subcellular distribution of these proteins is quite dissimilar (22): ACBP is almost exclusively cytosolic; L-FABP is both cytosolic and associated with microsomes; SCP-2 is highly enriched (8-fold) in peroxisomes and less so (2-fold) in endoplasmic reticulum (43, 44). This suggests that ACBP, while not exclusively functioning in fatty acyl-CoA binding, may regulate fatty acyl-CoA function in different intracellular site(s).
The structural and dynamic data obtained using ACBP fluorescence characteristics in the present studies also provided several unique insights on the nature of this protein and its interaction with the most prevalent 18-carbon fatty acids.
First, ACBP appears unique among the cytosolic lipid binding proteins with regard to in its spectroscopic distribution of aromatic amino acid residues. Careful analysis revealed the presence of a strong Tyr emission component in ACBP emission spectra despite the presence of Trp residues in the protein. In contrast, no resolvable Tyr component was observed in emission spectra of another fatty acyl-CoA binding protein, intestinal fatty acid binding protein (I-FABP) (27), even though I-FABP, like ACBP, also contains four Tyr and two Trp amino acid residues. ACBP Tyr emission was observable because of the favorable conditions preventing excitation energy transfer within Tyr-Trp donor-acceptor pairs, internal Tyr fluorescence quenching by peptide bonds, hydrogen bond formation, or photochemical reactions in Tyr excited singlet state (45). These findings allowed resolution of new structure and molecular dynamics features of apo- and holo-ACBP.
Second, the above observations were used to show that the intrinsic Tyr fluorescence could be used to determine binding of the 18-carbon oleoyl-CoA to this protein. The mechanism of Tyr, but not Trp, fluorescence quenching upon oleoyl-CoA binding to ACBP was dynamic in nature as indicated by a perfect correlation between the level (~2-fold) of ACBP fluorescence decrease (Fig. 4A) and the decrease (~2-fold) of the average fluorescence lifetime value (275/313 excitation, Table II).
Third, ACBP Trp residues were at least partially spectroscopically resolvable and, along with bound ligand rotational data, were used to infer that native rat liver ACBP was ellipsoidal with semiaxes of 15 and 9 Å.
Fourth, the rotational dynamics of ACBP showed that ligand binding induced a significant change in tertiary structure of this protein as evidenced by the decrease of both short and long axis lengths by almost 2 Å. Interestingly, no alterations in ACBP secondary structure induced by the bound ligand were detectable by either circular dichroic spectra of rat ACBP3 or by NMR spectroscopy of bovine recombinant ACBP (46). Similar observations of ligand-sensitive tertiary structure lability detectable by fluorescence techniques were also reported for other fatty acyl-CoA binding proteins, SCP-2 (29), L-FABP (27, 39), and I-FABP (27).
The role and significance of such protein conformational lability in protein-regulated lipid metabolic reactions, cell signaling (17-19), interactions with DNA/DNA binding proteins (20, 21), molecular recognition, or protein-protein interaction remain to be evaluated. In the latter cases, for example, it was recently shown that L-FABP isoforms possessing distinct secondary and tertiary structures (39) differentially modulated microsomal glycerol-3-phosphate acyltransferase and lysophosphatidic acyltransferase activities (41). Also, the holo-L-FABP isoform with bound fatty acid was a more potent stimulator of oleoyl-CoA incorporation into the phospholipids, as compared with its apo-form (41). Similar observations were made with another member of the fatty acid binding protein superfamily, the cellular retinol binding protein. The apo- and holo-forms of cellular retinol binding protein were selectively recognized by lecithin-retinol acyltransferase, resulting in differential modulation of enzyme activity (47).
In summary, the data presented new insights on the binding affinity, specificity, and potential function of ACBP in fatty acyl-CoA utilization. Furthermore, unique aspects of rat liver ACBP fluorescence emission allowed examination of the structural characteristics of apo- and holo-ACBP in solution, as well as the molecular dynamics of bound fatty acyl-CoA ligand measured in the nanosecond time range. This information significantly contributes to our understanding of ACBP structure and function(s) and can be used for a selective modification of ACBP functional activity by site-directed mutagenesis.
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FOOTNOTES |
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* This work was supported in part by USPHS Grant DK41402 from the National Institutes of Health.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 Physiology
and Pharmacology, Texas A & M University, TVMC, College Station, TX
77843-4466. Tel.: 409-862-1433; Fax: 409-862-4929; E-mail: fschroeder{at}cvm.tamu.edu.
1 The abbreviations used are: ACBP, acyl-CoA binding protein; L-FABP, liver fatty acid binding protein; SCP-2, sterol carrier protein-2; I-FABP, intestinal fatty acid binding protein; CoASH, coenzyme A, 9Z,11E,13E,15Z-octadecatetraenoyl coenzyme A, cis-parinaroyl-CoA.
2 T. H. Cho and F. Schroeder, unpublished observations.
3 A. Frolov and F. Schroeder, unpublished results.
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REFERENCES |
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