Salt Modulates the Stability and Lipid Binding Affinity of the Adipocyte Lipid-binding Proteins

• DOI: 10.1074/jbc.m304955200
• Publication Date: 2003-08-22T19:52:30Z
• Authors: Allyn J. Schoeffler; Carmen R. Ruiz; Allison M. Joubert; Xuemei Yang; Vince J. LiCata
• Keywords: Protein–lipid interaction

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