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• W2079224636 abstract "Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its ΔG of unfolding by 3–5 kcal/mol. At 37 °C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP. Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its ΔG of unfolding by 3–5 kcal/mol. At 37 °C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP. Adipocyte lipid-binding protein (ALBP or aP2) 1The abbreviations used are: ALBP, adipocyte lipid-binding protein; iLBP, intracellular lipid-binding protein; apo-ALBP, ALBP without bound lipid; holo-ALBP, ALBP with bound lipid; ANS, 1-anilinonaphthalene-8-sulfonate.1The abbreviations used are: ALBP, adipocyte lipid-binding protein; iLBP, intracellular lipid-binding protein; apo-ALBP, ALBP without bound lipid; holo-ALBP, ALBP with bound lipid; ANS, 1-anilinonaphthalene-8-sulfonate. is a member of the intracellular lipid-binding protein (iLBP) family, also known collectively as the intracellular fatty acid-binding proteins. Members of the iLBP family are found in a number of mammalian tissues, including liver, adipose, heart, brain, intestinal, and epithelial tissues (for reviews, see e.g. Refs. 1Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar, 2Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar, 3Stewart J.M. Cell. Mol. Life Sci. 2000; 57: 1345-1359Crossref PubMed Scopus (31) Google Scholar). As its name suggests, ALBP is found predominantly in adipocytes, where it constitutes 1–5% of the total soluble protein. In addition, ALBP is found in macrophages, where it has been linked to the development of atherosclerosis (4Layne M.D. Patel A. Chen Y.-H. Rebel V.I. Carvajal I.M. Pellacani A. Ith B. Zhao D. Schreiber B.M. Yet S.-F. Lee M.-E. Storch J. Perrella M.A. FASEB J. 2001; 15: 2733-2735Crossref PubMed Scopus (55) Google Scholar, 5Perrella M.A. Pellacani A. Layne M.D. Patel A. Zhao D. Schreiber B.M. Storch J. Feinberg M.W. Hsieh C.M. Haber E. Lee M.E. FASEB J. 2001; 15: 1774-1776Crossref PubMed Scopus (35) Google Scholar). ALBP has also been implicated in the development of type II diabetes (6Hotamisligil G.S. Johnson R.S. Distel R.J. Ellis R. Papaioannou V.E. Spiegelman B.M. Science. 1996; 274: 1377-1379Crossref PubMed Scopus (647) Google Scholar). Like most iLBPs, ALBP can bind a number of fatty acids of at least 14 carbons as well as other hydrophobic ligands with carboxylate or sulfonate moieties in its large, water-filled cavity (reviewed in Ref. 4Layne M.D. Patel A. Chen Y.-H. Rebel V.I. Carvajal I.M. Pellacani A. Ith B. Zhao D. Schreiber B.M. Yet S.-F. Lee M.-E. Storch J. Perrella M.A. FASEB J. 2001; 15: 2733-2735Crossref PubMed Scopus (55) Google Scholar). ALBP also tightly binds the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) (7Kane C.D. Bernlohr D.A. Anal. Biochem. 1996; 233: 197-204Crossref PubMed Scopus (85) Google Scholar, 8Ory J.J. Banszak L.J. Biophys. J. 1999; 77: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). ANS binds specifically in the ALBP lipid binding cavity (see Fig. 1), is a competitive inhibitor of fatty acid binding, and produces a large increase in fluorescence upon binding (7Kane C.D. Bernlohr D.A. Anal. Biochem. 1996; 233: 197-204Crossref PubMed Scopus (85) Google Scholar, 8Ory J.J. Banszak L.J. Biophys. J. 1999; 77: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Members of the iLBP family share remarkably similar tertiary structures. The iLBP fold consists of a 10-strand antiparallel β-barrel and a helix-turn-helix cap (see Fig. 1). They all possess a ligand binding cavity of ∼1000 Å3 located in the top half of the β-barrel (4Layne M.D. Patel A. Chen Y.-H. Rebel V.I. Carvajal I.M. Pellacani A. Ith B. Zhao D. Schreiber B.M. Yet S.-F. Lee M.-E. Storch J. Perrella M.A. FASEB J. 2001; 15: 2733-2735Crossref PubMed Scopus (55) Google Scholar). In contrast, the amino acid sequence homology between family members ranges from 23 to 69%, with 39 highly conserved residues (4Layne M.D. Patel A. Chen Y.-H. Rebel V.I. Carvajal I.M. Pellacani A. Ith B. Zhao D. Schreiber B.M. Yet S.-F. Lee M.-E. Storch J. Perrella M.A. FASEB J. 2001; 15: 2733-2735Crossref PubMed Scopus (55) Google Scholar). In addition, iLBPs each have distinct surface charge potentials (10LiCata V.J. Bernlohr D.A. Proteins. 1998; 33: 577-589Crossref PubMed Scopus (40) Google Scholar). ALBP has a nearly hemispherically distributed surface charge potential: mostly positively charged on the top and mostly negatively charged on the bottom (10LiCata V.J. Bernlohr D.A. Proteins. 1998; 33: 577-589Crossref PubMed Scopus (40) Google Scholar). The varied charge topologies of the different iLBPs suggest that surface charge may play an important role in differentiating them in vivo. The role that surface charge plays in iLBP function has to date been predominantly characterized in a series of studies by Storch and associates (11Wootan M.G. Bernlohr D.A. Storch J. Biochemistry. 1993; 32: 8622-8627Crossref PubMed Scopus (59) Google Scholar, 12Wootan M.G. Storch J. J. Biol. Chem. 1994; 269: 10517-10523Abstract Full Text PDF PubMed Google Scholar, 13Smith E.R. Storch J. J. Biol. Chem. 1999; 274: 35325-35330Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), which examine the interactions of iLBPs with lipid vesicles. These studies have demonstrated that ALBP transfers a fluorescent fatty acid to negatively charged lipid membranes more rapidly than to neutral or positively charged membranes (12Wootan M.G. Storch J. J. Biol. Chem. 1994; 269: 10517-10523Abstract Full Text PDF PubMed Google Scholar), that the transfer involves direct collision/interaction of ALBP and the vesicle (11Wootan M.G. Bernlohr D.A. Storch J. Biochemistry. 1993; 32: 8622-8627Crossref PubMed Scopus (59) Google Scholar, 12Wootan M.G. Storch J. J. Biol. Chem. 1994; 269: 10517-10523Abstract Full Text PDF PubMed Google Scholar), and that salt attenuates the transfer reaction (12Wootan M.G. Storch J. J. Biol. Chem. 1994; 269: 10517-10523Abstract Full Text PDF PubMed Google Scholar). Neutralizing the surface lysines of ALBP by acetylation also inhibits the ability of ALBP to form complexes with anionic vesicles (13Smith E.R. Storch J. J. Biol. Chem. 1999; 274: 35325-35330Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These studies demonstrate that electrostatic attraction is directly involved in the mediation of ALBP-membrane interactions. In addition to the surface electrostatic topologies and the ALBP-membrane interaction studies mentioned above, a variety of other recent studies support the significance of electrostatic effects in iLBP-lipid and ANS-protein interactions. 1) Mutation of charged residues in the binding cavity of intestinal fatty acid-binding protein can change the lipid binding specificity of the protein (14Jakoby M.G. Miller K.R. Toner J.J. Bauman A. Cheng L. Li E. Cistola D.P. Biochemistry. 1993; 32: 872-878Crossref PubMed Scopus (82) Google Scholar). 2) The binding of ANS to intestinal fatty acid-binding protein has been observed to be dependent on ionic strength (I) at low NaCl concentrations, and this observation was attributed to an electrostatic screening effect (15Kirk W.R. Kurian E. Prendergast F.G. Biophys. J. 1996; 70: 69-83Abstract Full Text PDF PubMed Scopus (102) Google Scholar). 3) Recent studies of “nonspecific” ANS surface binding to a series of different proteins using titration calorimetry concluded that most ANS molecules bind predominantly via electrostatic association and to a lesser extent via hydrophobic interactions (16Matulis D. Lovrien R. Biophys. J. 1998; 74: 422-429Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). 4) In ANS displacement assays, ALBP has been shown to bind retinoic acid, which has a carboxylate group, with moderate affinity, whereas the protein exhibits negligible affinity for retinol (7Kane C.D. Bernlohr D.A. Anal. Biochem. 1996; 233: 197-204Crossref PubMed Scopus (85) Google Scholar). Thus, whereas in one sense it might seem unusual to characterize electrostatic effects on interactions that are frequently considered to be predominantly hydrophobic, it is becoming increasingly clear that electrostatic effects are a major regulator of lipid-protein interactions. This has led us to investigate the effects of salt on ALBP. The results demonstrate and quantitate the regulatory effects of salt on the function and stability of ALBP. Purification of ALBP—ALBP was purified as described previously (17Xu Z. Buelt M.K. Banaszak L.J. Bernlohor D.A. J. Biol. Chem. 1991; 226: 14367-14370Abstract Full Text PDF Google Scholar, 18Simpson M.A. Bernlohr D.A. Biochemistry. 1998; 37: 10980-10986Crossref PubMed Scopus (21) Google Scholar). The pRSET plasmid for overexpression of murine ALBP was a gift from Dave Bernlohr at the University of Minnesota. An extinction coefficient of 15,500 m–1 cm–1 was used to determine protein concentrations (19Matarese V. Bernlohr D.A. J. Biol. Chem. 1988; 263: 14544-14551Abstract Full Text PDF PubMed Google Scholar). Protein was stored at –70 °C until use. Chemical Denaturations—For chemical denaturation studies, ALBP was first dialyzed extensively against appropriate buffers (10 mm potassium phosphate at pH 7.5, with varying KCl concentrations from 0 to 2 m). Urea stocks were deionized by stirring with AG 501-X8 deionizing resin (5 g/100 ml of solution, obtained from Bio-Rad) for 1 h in water. Urea concentrations were then determined by refractive index as described by Pace (20Pace C.N. Methods Enzymol. 1986; 14: 266-280Crossref Scopus (2412) Google Scholar), and the stocks were incorporated into the appropriate buffers. Stepwise chemical denaturations were performed by incubating individual aliquots of protein at 0.1–0.2 mg/ml with buffered urea for 1 h, well past the time required to reach equilibrium (data not shown). For denaturations of ALBP with ANS bound, 100 μm ANS was included in all experimental buffers. This concentration of ANS is saturating at all salt concentrations examined. Denaturation was monitored by scanning each sample in an Aviv model 202 circular dichroism spectrophotometer from 225 to 213 nm in a quartz cuvette with a 0.2-cm path length. Reversibility of ALBP unfolding was determined at high (1 m) and low (50 mm) salt concentrations by incubating ALBP in denaturing levels of urea plus salt for 1 h, followed by dialysis to remove the urea. Redenaturation was then performed, and full reversibility (recovery of the same ΔG unfolding) was obtained. Data Analysis of Denaturation Curves—The raw CD signal (in millidegrees) of each sample at 216, 217, and 218 nm was transformed into molar ellipticity (Δϵ) (21Johnson Jr., W.C. Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 145-166Crossref PubMed Scopus (504) Google Scholar), and denaturation curves at each wavelength were analyzed using the nonlinear form of the linear extrapolation method (22Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1599) Google Scholar). Δε=(ΔεN+mN[D])+(ΔεU+mU[D])e-(ΔGN→U0/RT+mG[D]/RT)1+e-(ΔGN→U0/RT+mG[D]/RT)(Eq. 1) Here Δϵ represents the molar ellipticity at a given wavelength (the dependent variable), [D] is the molar denaturant concentration (the independent variable), ΔϵN is the y intercept of the native state base line, m N is the slope of the native state base line, ΔϵU is the y intercept of the unfolded state baseline, m U is the slope of the unfolded state base line, ΔG0N→U is the extrapolated free energy of unfolding in the absence of denaturant, R is the gas constant, T is the temperature in Kelvin, and m G (the “m value”) is the slope of the calculated dependence of ΔG on [D]. Data were fit using the program KaleidaGraph (Synergy Software, Inc.). ANS Binding—The binding of ANS to ALBP was monitored using a FluoroMax-2 fluorometer. ANS concentrations in ethanol were determined using the extinction coefficient 7800 m–1 cm–1 (Molecular Probes, Inc., Eugene, OR). ANS in buffer plus 1% ethanol was titrated into protein (in 10 mm potassium phosphate, pH 7.5, containing concentrations of KCl as noted) and using protein concentrations near 0.05 μm. Samples were incubated for 2 min with stirring, and then fluorescence was monitored with 5-nm slits and excitation and emission wavelengths of 369 and 470 nm. Fluorescence was corrected for volume increase during the titration. A corresponding titration of the background fluorescence of ANS titrated into buffer was collected and subtracted from each individual binding titration. The resulting fluorescence titrations were analyzed using a single-site binding isotherm, F=(Fmax*[ANS]/Kd)/(1+[ANS]/Kd)(Eq. 2) where F max is the maximum fluorescence obtained upon binding, [ANS] is the concentration of ANS, and Kd is the dissociation constant of ANS from ALBP. This equation assumes that the total concentration of ANS is negligibly different from the free ANS concentration, which is true when Kd ≫ [ALBP]. Fits of the isotherms with the lowest (tightest) Kd values using a quadratic solution of the binding polynomial, which does not make this assumption (23Heyduk T. Lee J.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1744-1748Crossref PubMed Scopus (209) Google Scholar, 24Inglese J. Blatchly R.A. Benkovic S.J. J. Med. Chem. 1989; 32: 937-940Crossref PubMed Scopus (42) Google Scholar, 25Datta K. LiCata V.J. J. Biol. Chem. 2003; 278: 5694-5701Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), yield the same Kd values within error and verify the applicability of Equation 2 to these data. The lowest (tightest) Kd values in this study, at the lowest salt concentrations, are ∼10 times higher than the [ALBP]. Data were fit using the program KaleidaGraph. It should be noted that the ANS binding activity of ALBP is extremely sensitive to storage and preparation conditions, particularly to being stored in dilute form. In all of our experiments, protein was stored at –70 °C at a concentration greater than 3 mg/ml and thawed and diluted immediately before use. Even short periods (e.g. 16 h) of incubation in dilute (<10 μm) solution at 4 °C result in a significant loss of binding affinity. In addition, the use of nitrocellulose filters for buffer preparation results in interference in the fluorescence assay. Neither of these problems affects the stability of ALBP. Salt Linkage Analysis—Linked ion release upon binding of ANS to ALBP was calculated using a basic linkage relationship (26Wyman J. Adv. Protein Chem. 1964; 19: 223-286Crossref PubMed Scopus (1305) Google Scholar, 27Lohman T.M. Mascotti D.P. Methods Enzymol. 1992; 212: 400-424Crossref PubMed Scopus (142) Google Scholar); e.g. for KCl, the relationship is as follows. {∂ln1/Kd}/{∂ln[KCl]}=Δnions=ΔnK++ΔnCl-(Eq. 3) Thus, a plot of ln(1/K dANS) versus ln [KCl] will have a slope equivalent to the net number of ions that are bound or released when ANS binds. Direct Ligand Displacement—To examine the direct displacement of ANS by salt, KCl was titrated into ANS-bound ALBP, and the decrease in fluorescence as ANS was released was recorded. A background titration of buffer added to ANS-bound ALBP was subtracted from the salt displacement data. The data were fit with a simple inverse isotherm, YI={[KCl]/IC50}/{1+[KCl]/IC50}(Eq. 4) where Y I is the normalized fluorescence, and IC50 is the concentration of salt at 50% inhibition. Stopped Flow Kinetic Analysis of ANS Binding to ALBP—Kinetics experiments were performed using a Biologic SF3 stopped flow interfaced with an ISS fluorometer. ALBP and ANS were rapidly mixed, and ANS fluorescence was monitored with excitation at 369 nm and emission at 470 nm. The assay buffer was 10 mm potassium phosphate, pH 7.5, 1% ethanol, and either 50 mm KCl or 1 m KCl. The resulting kinetic association curves were fit to the single exponential equation, F=(Fmax-F0)·(1-e-kobs(t-toffset))+F0(Eq. 5) where F is the fluorescence (dependent variable), (F max – F 0) is the amplitude of the transition, k obs is the time constant of association, t is the time after the completion of injection (independent variable), t offset is the x axis offset, which accounts for mixing time and the dead time of the instrument, F 0 is the initial fluorescence before binding, and F max is the maximum fluorescence upon completion of binding. All parameters were allowed to float except F 0, which was determined by observing the fluorescence of ANS in buffer and was then fixed during fitting. The association of ANS with ALBP is very fast, so experiments were conducted at 6 °C to minimize loss of data during the dead time (∼3 ms). For each concentration of ANS + ALBP, 5–12 shots over two different time scales (100 ms and 1 s) were averaged and collated. Using this technique, ∼50% of the full extent of reaction can be captured (less for faster processes, more for slower processes), which affords excellent precision in the determination of k obs. Determined rates are not affected by the collection time scale, but averaging and obtaining high density overlapping data throughout the complete course of the reaction significantly improves the precision of the fits. Kinetic curves were fit to both single and double exponential functions with the result that all kinetic curves were judged to be single exponential curves. It should be noted that the sensitivity of ALBP to storage conditions, described under “ANS Binding,” also appears to pertain to the kinetic behavior of the protein. Carefully handled and stored protein consistently produced well behaved single exponential kinetic curves. For determination of the association (k on) and dissociation (k off) rate constants, the relaxation time constants were plotted as a function of the sum of the equilibrium concentrations of ANS plus ALBP (28Bernasconi C.F. Relaxation Kinetics. Academic Press, Inc., 1976: 11-13Crossref Google Scholar). Equilibrium concentrations of ANS and ALBP were determined by an iterative fitting process (28Bernasconi C.F. Relaxation Kinetics. Academic Press, Inc., 1976: 11-13Crossref Google Scholar). Briefly, 1/τ (1/τ = k obs) is first plotted as a function of total [ANS], and the ratio between the intercept (k off) and the slope (k on) of this plot is used to calculate a dissociation constant (Kd = k off/k on). This preliminary Kd is used along with the known total concentrations of ANS and ALBP in each sample to calculate new equilibrium concentrations for ANS and ALBP, which are then used to generate a new plot of 1/τ versus [ANS]eq + [ALBP]eq. Seven to ten iterations of this procedure were sufficient to achieve excellent convergence. Analytical Ultracentrifugation—Sedimentation equilibrium experiments were performed in 10 mm potassium phosphate, pH 7.5, at low (50 mm) and high (1 m) KCl concentrations, in a Beckman Optima XL-A analytical ultracentrifuge. The sample and reference sectors of Epon charcoal-filled double-sector cells were loaded, respectively, with 110 μl of unligated or ANS-bound ALBP in low or high salt buffer and 125 μl of the corresponding buffer. For the runs in the presence of ANS, the reference sector also contained ANS, the ANS concentration was ∼100 μm, and the samples contained 1% ethanol. Runs were performed at 20 °C and 25,000 rpm for ∼24 h. Absorbance was measured at 279 nm for unligated ALBP and 281 nm for ANS-bound ALBP. The ALBP concentration was 0.2 mg/ml. Data were analyzed using the Origin equilibrium analysis program in the Beckman analysis software package. Values of the partial specific volume of ALBP and the densities of the buffer solutions at 20 °C were calculated using the computer program SEDNTERP (available on the World Wide Web at biochem.uthscsa.edu/auc/software). Fitting data to models of higher oligomeric complexity did not reveal any contaminants or additional equilibria and confirmed that the data depict an ideal single species in solution. Salt Significantly Stabilizes ALBP—Native ALBP displays a CD spectrum typical of a protein predominantly consisting of β-sheet. Fig. 2A shows the change in the CD spectrum as urea unfolds the protein. The largest spectral changes occur in the β-sheet trough around 217 nm, so for each denaturation the signals at wavelengths of 216, 217, and 218 nm were individually analyzed and averaged to obtain values for ΔG unfolding as described under “Experimental Procedures.” Fig. 2B shows representative denaturation curves for ALBP in different solution conditions. Urea denaturations of apo-ALBP at 25 °C as a function of added KCl reveal a significant (3 kcal/mol) salt-induced stabilization of the protein as the salt concentration is increased from 0 to 2 m (Table I). The salt stabilization effect on apo-ALBP appears to be mostly saturated at KCl concentrations above 250 mm. Measurements of the ΔG unfolding at 37 °C in the presence and absence of 2 m KCl also show a significant (5.1 kcal/mol) salt-induced stabilization of ALBP. Thus, at 37 °C, the addition of 2 m salt more than doubles the stability of the protein. This is an extremely large salt-induced protein stabilization.Table IΔGunfolding of ALBP with increasing KCl (in kcal/mol)[KCl]apo-ALBP, 25 °Capo-ALBP, 37 °CANS-bound ALBP, 25 °CaIn all ANS-bound ALBP denaturations, the concentration of ANS was 100 μm.mm04.8 ± 0.84.0 ± 1.09.7 ± 2.0504.8 ± 0.68.7 ± 1.12506.8 ± 0.77.1 ± 1.05006.7 ± 0.76.7 ± 0.910006.7 ± 0.46.8 ± 0.720007.8 ± 0.99.1 ± 1.4a In all ANS-bound ALBP denaturations, the concentration of ANS was 100 μm. Open table in a new tab Salt Appears to Destabilize Lipid-bound ALBP—In contrast to the effect of salt on apo-ALBP, ANS-bound ALBP appears to be destabilized by KCl. At 0 mm KCl, ANS-bound ALBP has a free energy of unfolding of 9.7 kcal/mol. Additional stabilization relative to the apoprotein is expected from the contribution of the ANS binding energy. The binding of any ligand to a protein will stabilize it. However, instead of exhibiting a pattern of stabilization paralleling the apoprotein (offset by the added ΔG binding of ANS), the holoprotein appears to become destabilized as salt is added. In general, the denaturation curves are less well behaved at high ANS concentrations, and this leads to higher propagated errors on the determined free energies. This situation results in significant overlap of the fitted ΔG unfolding values in the presence of bound ANS (Table I). However, although the error envelopes overlap, the low salt data cannot be fit using the high salt value of ΔG unfolding (6.8 kcal/mol), and the high salt data cannot be fit using the low salt value of ΔG unfolding (9.7 kcal/mol). Furthermore, the data clearly and statistically significantly indicate that unlike the case for apo-ALBP, the ΔG unfolding for holo-ALBP is not increasing with added salt. As discussed below, direct lipid binding experiments were conducted, which demonstrate that a major source (but not the sole source) of this effect is the salt-induced displacement of ANS from the protein. Thermodynamic linkage calculations indicate that this effect is coupled with a salt-induced increase of the affinity of ANS for the denatured state of ALBP. The combination of these two effects produces the observed results and results in the observed convergence of the stabilization energies for the apo- and holoproteins at higher salt concentrations (see “Thermodynamic Linkages among Stability, Ligand Binding, and Salt”). All of the unfolding data fit well to a two-state denaturation model (Equation 1). It is, however, possible that the observed stabilization is the result of a salt-induced shift between a two-state unfolding process and a three-state unfolding process (which would have a lower apparent ΔG unfolding when analyzed as a two-state process). If this were true, however, the shift for the apoprotein would have to be three-state to two-state with added salt, whereas the shift for holoprotein would have to be two-state to three-state. Salt Inhibits Lipid Binding to ALBP and Causes Lipid Release—Equilibrium binding of ANS to ALBP was measured using the intrinsic fluorescence increase of ANS upon binding. ANS binds specifically in the lipid binding cavity of ALBP (4Layne M.D. Patel A. Chen Y.-H. Rebel V.I. Carvajal I.M. Pellacani A. Ith B. Zhao D. Schreiber B.M. Yet S.-F. Lee M.-E. Storch J. Perrella M.A. FASEB J. 2001; 15: 2733-2735Crossref PubMed Scopus (55) Google Scholar, 8Ory J.J. Banszak L.J. Biophys. J. 1999; 77: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and competes directly with the binding of other lipids (7Kane C.D. Bernlohr D.A. Anal. Biochem. 1996; 233: 197-204Crossref PubMed Scopus (85) Google Scholar). Fig. 3A shows representative titrations at different KCl concentrations. Binding as a function of increased salt reveals that KCl weakens the association between ANS and ALBP. The Kd of ANS binding to ALBP increases from 0.48 to 4.4 μm upon increasing the KCl concentration from 50 to 750 mm (see Fig. 3 and Table II).Table IIANS-ALBP binding as a function of KCl[KCl]KdΔGmmμ mkcal/mol500.48 ± 0.148.62 ± 2.541501.37 ± 0.057.99 ± 0.292501.21 ± 0.438.07 ± 2.885002.27 ± 0.207.69 ± 0.677504.40 ± 0.077.30 ± 0.12 Open table in a new tab A thermodynamic linkage analysis of these data is also shown in Fig. 3. The slope of a plot of ln [salt] versus ln(1/Kd) provides an estimate of the net uptake or displacement of ions upon ligand binding (26Wyman J. Adv. Protein Chem. 1964; 19: 223-286Crossref PubMed Scopus (1305) Google Scholar, 27Lohman T.M. Mascotti D.P. Methods Enzymol. 1992; 212: 400-424Crossref PubMed Scopus (142) Google Scholar). Fig. 3 shows that there exists a thermodynamic linkage of ∼0.8 ions released upon binding of ANS. In other words, ANS binding causes the dissociation of essentially one monovalent ion from ALBP. Since thermodynamic linkages must be reciprocal, adding salt to holo-ALBP must also displace bound ligand. The salt-induced dissociation of ANS from ALBP was confirmed by titrating KCl into ANS-bound ALBP and observing the concomitant loss of fluorescence as the ANS dissociated (Fig. 3C). Note that ANS still binds to ALBP at high salt (Table II and data not shown), so Fig. 3C does not represent 100% displacement of ANS from the protein but shows a salt-induced equilibrium shift at one concentration of ANS. Analysis of the salt-induced displacement of ANS with a simple inverse isotherm (Equation 2), representing competitive inhibition, yields an IC50 value of 172 mm KCl. Salt Effects on the Kinetics of ANS-ALBP Binding—The kinetics of ANS association with ALBP were monitored by stopped flow fluorescence spectroscopy over a range of ANS concentrations at 50 mm and 1 m KCl. Fig. 4A shows representative kinetic curves at" @default.
• W2079224636 created "2016-06-24" @default.
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• W2079224636 creator A5013680328 @default.
• W2079224636 creator A5052957147 @default.
• W2079224636 creator A5059350064 @default.
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• W2079224636 date "2003-08-01" @default.
• W2079224636 modified "2023-09-27" @default.
• W2079224636 title "Salt Modulates the Stability and Lipid Binding Affinity of the Adipocyte Lipid-binding Proteins" @default.
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