Mutants of Rat Intestinal Fatty Acid-binding Protein Illustrate the Critical Role Played by Enthalpy-Entropy Compensation in Ligand Binding
1997; Elsevier BV; Volume: 272; Issue: 27 Linguagem: Inglês
10.1074/jbc.272.27.16737
ISSN1083-351X
AutoresGary V. Richieri, Pamela J. Low, Ronald T. Ogata, Alan M. Kleinfeld,
Tópico(s)Receptor Mechanisms and Signaling
ResumoSite-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity. Site-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity. Fatty acid-binding proteins (FABPs) 1The abbreviations used are: FABP, fatty acid-binding protein; I-FABP, intestinal FABP; ADIFAB, acrylodated I-FABP; WT, wild type; FFA, free fatty acids; AA, arachidonate (20:4); LA, linoleate (18:2); LNA, linolenate (18:3); SA, stearate (18:0); OA, oleate (18:1); PA: palmitate (16:0). are approximately 15-kDa cytosolic proteins that may play important roles in fatty acid (FA) trafficking (1Bass N.M. Int. Rev. Cytol. 1988; 111: 143-184Crossref PubMed Scopus (234) Google Scholar, 2Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar, 3Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar). X-ray crystallography reveals that the FA binding site is an internal cavity in the protein (4Sacchettini J.C. Gordon J.I. Banaszak L.J. J. Mol. Biol. 1989; 208: 327-339Crossref PubMed Scopus (290) Google Scholar, 5Cowan S.W. Newcomer M.E. Jones T.A. J. Mol. Biol. 1993; 230: 1225-1246Crossref PubMed Scopus (160) Google Scholar, 6Young A.C.M. Scapin G. Kromminga A. Patel S.B. Veerkamp J.H. Sacchettini J.C. Structure. 1994; 2: 523-534Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 7Thompson J. Winter N. Terwey D. Bratt J. Banaszak L. J. Biol. Chem. 1997; 272: 7140-7150Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 8LaLonde J.M. Bernlohr D.A. Banaszak L.J. Biochemistry. 1994; 33: 4885-4895Crossref PubMed Scopus (71) Google Scholar). Crystal structures of rat intestine FABP (I-FABP) and its complex with FA show that the hydrocarbon chain of the FA interacts directly with about 19 amino acid resides and several bound waters within this cavity (2Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar, 4Sacchettini J.C. Gordon J.I. Banaszak L.J. J. Mol. Biol. 1989; 208: 327-339Crossref PubMed Scopus (290) Google Scholar, 9Eads J. Sacchettini J.C. Kromminga A. Gordon J.I. J. Biol. Chem. 1993; 268: 26375-26385Abstract Full Text PDF PubMed Google Scholar). Binding of FA to FABP involves desolvation of the FA followed by insertion into the binding cavity. The net free energy for these steps for wild type I-FABP is approximately −10 kcal/mol (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Kurian E. Kirk W.R. Prendergast F.G. Biochemistry. 1996; 35: 3865-3874Crossref PubMed Scopus (46) Google Scholar) and is predominately enthalpic (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Kurian E. Kirk W.R. Prendergast F.G. Biochemistry. 1996; 35: 3865-3874Crossref PubMed Scopus (46) Google Scholar, 12Jakoby 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). To understand how the amino acid residue-FA interactions revealed by the crystal structure contribute to the energy of binding we have used site-specific mutagenesis to alter amino acid residues within the binding cavity. Most mutagenesis studies aimed at understanding ligand binding interactions have relied on changes in affinity to determine which residues play important roles in the active site (13Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1783) Google Scholar, 14Mynarcik D.C. Yu G.Q. Whittaker J. J. Biol. Chem. 1996; 271: 2439-2442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, the affinity is related to the free energy change (K d = (e − (ΔG 0/RT))) and, through ΔG 0 = ΔH 0 −TΔS 0, to the enthalpy and entropy changes of the binding reaction. 2Although the binding constants are given as dissociation constants (K d ), the thermodynamic parameters throughout the paper are for the association reaction as in our previous studies (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The parameters for association are simply the negative of those for dissociation. Because mutations alter both enthalpic and entropic contributions to ligand binding, the changes in the underlying molecular interactions may not be correlated with changes in affinity (15Sturtevant J.M. Curr. Opin. Struct. Biol. 1994; 4: 69-78Crossref Scopus (83) Google Scholar, 16Dill K. J. Biol. Chem. 1997; 272: 701-704Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). In the present study, therefore, we have determined the free energy, enthalpy, and entropy changes of binding for each mutant interacting with long chain FAs. Mutants were constructed by extension of overlapping oligonucleotides, which together spanned restriction endonuclease sites in I-FABP, and insertion of the resulting double-stranded DNA as described (17Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1996; 271: 11291-11300Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Mutant and WT proteins were expressed in the pET/BL21 system as described (18Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar). Protein for all mutants except E51A and F93A were isolated from cell lysates. E51A and F93A were expressed as inclusion bodies and were solubilized by denaturation in 4 m GdnHCl followed by renaturation by dialysis against a buffer consisting of 10 mm HEPES, 150 mm NaCl, 5 mm KCl, and 1 mmNaHPO4, at pH 7.4. This buffer was also used in all the binding measurements. Protein purification and delipidation for all proteins was done as described (18Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar). ADIFAB was prepared from acrylodan-derivatized I-FABP as described (19Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1992; 267: 23495-23501Abstract Full Text PDF PubMed Google Scholar) and is available from Molecular Probes, Eugene, OR. Measurements of the binding of FA to FABP were done by using ADIFAB fluorescence to monitor the binding of the sodium salts of the FA to each FABP at temperatures between 10 and 45 °C as described (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). For each combination of FA and FABP, a binding isotherm was measured for each temperature and in all cases showed a stoichiometry of 1 FA binding per FABP monomer. The temperature dependence of the K d values for each FA and FABP exhibited linear van't Hoff behavior, and ΔH 0 values were determined from the slopes of each of the van't Hoff plots. For the results shown in Figs. 2 and 3, ΔG 0 values were evaluated from theK d values measured at 25 °C, and −TΔS 0 was calculated as ΔG 0 − ΔH 0.Figure 3Thermodynamic parameter differences for Arg-106. Mutant − WT differences at 25 °C are shown for Gln and Ala substitutions at position 106. These measurements were done for the 4 FA used in Fig. 2 plus linolenate (18:3). The disparity between previous calorimetry measurements showing a 20-fold reduction in oleate binding to the R106Q mutant (12Jakoby 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), as compared with the 3-fold reduction observed in the present study, is likely the result of the inability of calorimetry to measure accurately the binding affinities of FA-FABP complexes (11Kurian E. Kirk W.R. Prendergast F.G. Biochemistry. 1996; 35: 3865-3874Crossref PubMed Scopus (46) Google Scholar, 23Cistola D.P. Kim K. Rogl H. Frieden C. Biochemistry. 1996; 35: 7559-7565Crossref PubMed Scopus (80) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To obtain information about the nature of the FA-FABP interaction we constructed 24 mutants of I-FABP and measured their binding to FA. We substituted Ala for 16 of the 19 residues interacting with the FA in the cavity (Fig. 1 A), as well as Gln at residue 106. We also investigated Ala substitutions of the more distal amino acid residues shown in Fig. 1 B. FA binding to each mutant was measured as a function of temperature using the fluorescent probe ADIFAB (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1992; 267: 23495-23501Abstract Full Text PDF PubMed Google Scholar). Dissociation constants (K d ) determined at 37 °C for binding of 6 long chain FA to the wild type protein and to each of the 24 mutants were found to range between 0.5 and 4500 nm (Table I and Fig. 3). These results show that substitution of single residues in I-FABP can result in proteins that bind with affinities ranging from 30-fold higher (Leu-72, Arg-106) to almost 30-fold lower (Met-18, Phe-68) than the wild type (WT) protein.Table IDissociation constants (K d ) for fatty acid binding to wild type and site-specific mutants of rat intestinal fatty acid-binding proteinFABP1-aWT is the wild type I-FABP, and single-letter designations are used to indicate the WT residue, amino acid sequence position, and the substituted amino acid residue for each mutant.PASAOALALNAAAWT2062790367200L72A10.528812R106A4124108L102A713122627Y117A524103520Q115A136194912760E51A1462383230244Y14A1482445121147W82A38256188155100F93A1916558310066Y70A301644177611322N11A321047151478383L78A271643243620572M21A533994227408424V60A451360182437624F17A562773211461205F47A8221804041168855F62A892713545215651162I23A94321865091109589D34A706524749313301590F55A1045424847813761515R126A14814835252012501550M18A1499336674816402300F68A510365878218345001890K d values were measured at 37 °C and are given in nm. Measurement standard deviations are approximately 10%.1-a WT is the wild type I-FABP, and single-letter designations are used to indicate the WT residue, amino acid sequence position, and the substituted amino acid residue for each mutant. Open table in a new tab K d values were measured at 37 °C and are given in nm. Measurement standard deviations are approximately 10%. From the temperature dependence of the K d values we determined the differences in enthalpy (ΔΔH 0), entropy (TΔΔS 0), and free energy (ΔΔG 0) of FA binding between each mutant and the WT protein. These results, arranged so that the ΔΔG 0 values for linoleate increase monotonically, are shown in Fig. 2 and demonstrate that changes in affinity, equivalent to ΔΔG 0values, are not correlated with ΔΔH 0 andTΔΔS 0. This lack of correlation results because ΔΔH 0 andTΔΔS 0 tend to compensate in these binding reactions. As a consequence, the mutation-induced changes in binding enthalpy and entropy are almost always larger than ΔΔG 0, so that relatively small mismatches in the enthalpy/entropy compensation can result in highly significant changes in binding affinity. A striking feature of these results is that the changes in affinity are not related uniquely to the changes in enthalpy and entropy. For example, although Ala substitutions for Leu-72, Arg-106, and Tyr-117 all result in substantial increases in affinity, the molecular interactions that generate these increases are different in each case. The increase in affinity for L72A is caused by an increase in ‖ΔH 0‖ with a smaller decrease in entropy, that for R106A is caused by a decrease in ‖ΔH 0‖ with a larger increase in entropy, and that for Y117A by both of these kind of changes, but generally with smaller magnitudes. At the other end of the scale, M18A and F68A have substantially lower affinities than the WT protein. This is achieved by quite large (>6 kcal/mol) decreases in ‖ΔH 0‖ with smaller increases in entropy for M18A but quite modest ( 5 kcal/mol). Several of the mutants illustrate that large enthalpy changes of either sign can occur even in the absence of apparent electrostatic interactions. For instance, an Ala substitution of Leu-102 results in an average 4.7 kcal/mol more favorable enthalpy while the enthalpy of the M18A mutant has a less favorable enthalpy by an average of about 6.5 kcal/mol. In contrast to both of these examples in which large changes in the underlying molecular interactions are reflected in large changes in binding, the Y14A mutant reveals almost no change in binding, but up to 6 kcal/mol of almost exactly compensating ΔΔH 0 andTΔΔS 0.Table IIThermodynamic parameter differences between site-specific alanine mutants of amino acid residues in direct contact with bound FA and the WT I-FABPΔΔG0ΔΔH 0−TΔΔS 0PAOALAAAPAOALAAAPAOALAAAF68A2.11.91.71.41.71.11.4−10.40.80.32.4M18A*1.71.81.21.47.27.55.16−5.5−5.7−3.9−4.6F55A1.31.311.14.23.42.71.5−2.9−2.1−1.7−0.4I23A1.31.10.80.33.23.30.3−3.9−1.9−2.20.54.2F62A1.20.80.70.80.6−0.5−1−1.10.61.31.71.9F17A10.70.5−0.24.63.82.6−2.5−3.6−3.1−2.12.3V60A0.80.40.30.41.4−1.30.4−0.1−0.61.7−0.10.5L78A0.40.30.30.3−2.9−3.6−4.3−5.23.33.94.65.5F93A*0.20.3−0.1−0.91.42.33.6−1.3−1.21−3.70.4Y70A*0.40.10.20.21.3−2.3−1.1−1.6−0.92.41.31.8W82A*0.80.5−0.1−0.541.51−0.5−3.2−1−1.10Y14A0.20.1−0.3−0.23.54.64.86−3.3−4.5−5.1−6.2Y117A*−0.5−1.2−1.3−1.5−0.5−2.4−0.20.301.2−1.1−1.8L102A−0.7−1.5−1.5−1.5−4.6−3.9−5.4−6.13.92.43.94.6R106A*−0.6−1.7−2−25.93.23.20.9−6.5−4.9−5.2−2.9L72A*−1.5−1.6−2−1.8−3.21.4−5.8−3.51.7−33.81.7—2-aValues in this row are estimates of the WT thermodynamic parameters (ΔG 0, ΔH 0, and TΔS 0) calculated as: −∑ΔΔG i 0, −∑ΔΔHi0, ∑TΔΔ i 0, respectively, wherei represents each of the mutants in column 1.−8.1−3.30.62.7−27.8−18.1−7.312.119.711.87.9−9.4*2-bValues in this row are sums of a subset of mutants indicated by *. These mutants were chosen using the crystal structure (4) to pick residues that are at well-separated locations and may therefore reflect greater independence.−0.51.84.15.1−16.1−11.2−5.8−0.315.6109.95.4WT2-cWT values are the measured ΔG 0, ΔH 0, and TΔS 0reported previously (10).−11.1−10.6−9.8−9.4−11.5−10.8−10.5−11.60.40.20.72.2WT-desol2-dValues in this row are WT − the contributions estimated previously for the desolvation step (ΔG water0) in binding (10).−1.0−0.6−0.7−0.2−11.5−10.8−10.5−11.610.510.29.811.4The thermodynamic functions are for association of the fatty acid-protein complex. All values are in kcal/mol, and ΔΔG 0 and TΔΔS 0are calculated at 25 °C.2-a Values in this row are estimates of the WT thermodynamic parameters (ΔG 0, ΔH 0, and TΔS 0) calculated as: −∑ΔΔG i 0, −∑ΔΔHi0, ∑TΔΔ i 0, respectively, wherei represents each of the mutants in column 1.2-b Values in this row are sums of a subset of mutants indicated by *. These mutants were chosen using the crystal structure (4Sacchettini J.C. Gordon J.I. Banaszak L.J. J. Mol. Biol. 1989; 208: 327-339Crossref PubMed Scopus (290) Google Scholar) to pick residues that are at well-separated locations and may therefore reflect greater independence.2-c WT values are the measured ΔG 0, ΔH 0, and TΔS 0reported previously (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).2-d Values in this row are WT − the contributions estimated previously for the desolvation step (ΔG water0) in binding (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Open table in a new tab The thermodynamic functions are for association of the fatty acid-protein complex. All values are in kcal/mol, and ΔΔG 0 and TΔΔS 0are calculated at 25 °C. If the thermodynamic parameter differences for each Ala substitution indicate the individual interaction energies between the FA and the amino acid residues for the WT protein, then the sum of the individual interactions should be comparable with the total binding energies for the WT protein. The energies of Fig. 2 and Table II reflect interactions only within the cavity, while the measured ΔG 0 values for binding include interactions involved in the desolvation step. A significant portion of the desolvation free energy is entropic (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and the last row of Table IIshows the thermodynamic parameters with this contribution removed. As Table II indicates, the sums of the thermodynamic contributions for all of the mutants are significantly different from the measured ΔG 0, ΔH 0, andTΔS 0 values. That these sums do not equal the actual binding energies is not surprising because the ΔΔG 0, ΔΔH 0, andTΔΔS 0 values are generally not independent (16Dill K. J. Biol. Chem. 1997; 272: 701-704Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) and the values for the mutants may not accurately reflect the interactions in the WT protein (15Sturtevant J.M. Curr. Opin. Struct. Biol. 1994; 4: 69-78Crossref Scopus (83) Google Scholar). Studies of the gene V protein of bacteriophage f1 suggest that additivity might be more accurate for well separated mutations (20Sandberg W.S. Terwilliger T.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8367-8371Crossref PubMed Scopus (63) Google Scholar). An improvement is obtained by restricting the summation for the I-FABP mutants to 7 well-separated residues out of the total 15 cavity residues (the asterisk row of TableII), but significant differences remain. In particular, the sums of the cavity enthalpies differ systematically with double bond number, although the measured binding enthalpy is virtually identical (−11 kcal/mol) for each of the 4 FA (Table II). This variation may reflect interactions internal to the FA that differ in the bound and free state, and this difference may be different for each of the FA (21Rich M.R. Biochim. Biophys. Acta. 1993; 1178: 87-96Crossref PubMed Scopus (69) Google Scholar) and would be consistent with a compensating entropy as discussed previously (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Thus accurate estimates of the FA's internal energy differences in the solvent and I-FABP bound states would help to determine whether summation over mutation-induced changes in thermodynamic parameters provides an accurate estimate of the total binding energetics within the cavity. Some of the substituted I-FABPs involved residues that are more distal to the FA binding site (Fig. 1 B). Perhaps the most interesting of these involves Arg-126, which appears to play a direct role in FA binding to all FABPs except intestine where it is more than 6.5 Å from the FA (3Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar). Nevertheless, as Fig. 2 shows, R126A has a substantial effect on binding due to large ΔΔH 0 andTΔΔS 0. Because very similar changes are produced by the D34A mutation (Fig. 2), and because Arg-126 forms an electrostatic interaction with Asp-34 (2Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar, 3Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar, 9Eads J. Sacchettini J.C. Kromminga A. Gordon J.I. J. Biol. Chem. 1993; 268: 26375-26385Abstract Full Text PDF PubMed Google Scholar), the large effect on FA binding by both of these mutations suggests that this electrostatic bond is important, perhaps for the conformation of the binding cavity. Relatively small ΔΔH 0 andTΔΔS 0 at distal locations can also profoundly affect ligand binding as illustrated by Ala mutations of Phe-47 and Phe-68, which together with Phe-62 form a barrier between the interior and exterior of the protein and are ≥4.5 Å from the FA at the carboxylate end (2Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar). These mutations result in substantial reductions in affinity and indeed the F68A mutation produced the largest ΔΔG 0 (∼1.9 kcal/mol) of all 24 mutants. These alterations of ΔG 0 are, however, achieved by relatively small (generally less than 2 kcal/mol) non-compensating changes in ΔH 0 andTΔS 0 (F68A is the only mutant not exhibiting enthalpy/entropy compensation). The quite small effect for the relatively neutral residue Asn-11, which is predicted to be involved in the initial binding step (2Sacchettini J.C. Gordon J.I. J. Biol. Chem. 1993; 268: 18399-18402Abstract Full Text PDF PubMed Google Scholar) but is >10 Å from the FA, demonstrates that not all residues affect FA binding. The ability of FABPs to discriminate among different FA is an important issue in FA metabolism. In general FA metabolism exhibits a high degree of FA specificity, distinguishing clearly among FA on the basis of chain length and saturation (for example, Ref. 22Cook H.W. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. 2nd Ed. Elsevier, Amsterdam1991: 141-169Google Scholar). Presumably, FA recognition by proteins occurs at various steps in FA metabolism. Although FA binding to FABPs does not reveal the kind of selectivity observed in cellular metabolism (18Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar, 19Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1992; 267: 23495-23501Abstract Full Text PDF PubMed Google Scholar), FABPs might provide a good model of how selectivity can be built into FA recognition. Unfortunately, most of the mutants generated little change in binding specificity, and much of the specificity apparent in Table I (for example, K d values for all mutants are in the order SA<OA<LA<LNA) is a reflection of FA solubility differences (10Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1995; 270: 15076-15084Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 18Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1994; 269: 23918-23930Abstract Full Text PDF PubMed Google Scholar,19Richieri G.V. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1992; 267: 23495-23501Abstract Full Text PDF PubMed Google Scholar). However, a few of the mutants such as R106A, W82A, Y117A, Y14A, F93A, and M21A have significant effects on binding specificity. In the case of Trp-82, for example, the Ala mutation results in a monotonic increase in binding with double bond number for the 18-carbon series of FA, producing a 10-fold increase in binding of linolenate (18:3) relative to stearate (18:0). The molecular details of how these alterations are achieved are unclear, although the predominance of aromatic residues within the I-FABP binding cavity and the frequent deviations of arachidonate's (20:4) thermodynamic parameters relative to the other FA raise the possibility that the aromatic double bond interaction may play a role in modulating FA binding specificity.
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