Enantiospecificity of chloroperoxidase-catalyzed epoxidation: biased molecular dynamics study of a cis-β-methylstyrene/chloroperoxidase-compound I complex

Molecular dynamics simulations of an explicitly solvated cis-β-methylstyrene/chloroperoxidase-Compound I complex are performed to determine the cause of the high enantiospecificity of epoxidation. From the simulations, a two-dimensional free energy potential is calculated to distinguish binding potential wells from which reaction to 1S2R and 1R2S epoxide products may occur. Convergence of the free energy potential is accelerated with an adaptive biasing potential. Analysis of binding is followed by analysis of 1S2R and 1R2S reaction precursor structures in which the substrate, having left the binding wells, places its reactive double bond in steric proximity to the oxyferryl heme center. Structural analysis of binding and reaction precursor conformations is presented. We find that 1), a distortion of Glu(183) is important for CPO-catalyzed epoxidation as was postulated previously based on experimental results; 2), the free energy of binding does not provide significant differentiation between structures leading to the respective epoxide enantiomers; and 3), CPO's enantiospecificity toward cis-β-methylstyrene is likely to be caused by a specific group of residues which form a hydrophobic core surrounding the oxyferryl heme center.     

Molecular dynamics and free energy studies of chirality specificity effects on aminobenzo[a]quinolizine inhibitors binding to DPP-IV

The aminobenzo[a]quinolizines were investigated as a novel class of DPP-IV inhibitors. The stereochemistry of this class plays an important role in the bioactivity. In this study, the mechanisms of how different configuration of three chiral centers of this class influences the binding affinity were investigated by molecular dynamics simulations, free energy decomposition analysis. The S configuration for chiral center 3* is decisive for isomers to maintain high bioactivity; the chirality effect of chiral center 2* on the binding affinity is largely dependent, while the S configuration for chiral center 2* is preferable to R configuration for the bioactivity gain; the effect of chiral center 11b* on the binding affinity is insignificant. The chirality specificity for three chiral centers is responsible for distinction of two van der Waals contacts with Tyr547 and Phe357, and of H-bonding interactions with Arg125 and Glu206. Particularly, the Arg125 to act as a bridge in the H-bonding network contributes to stable H-bonding interactions of isomer in DPP-IV active site.

Computational study on the selective inhibition mechanism of MS402 to the first and second bromodomains of BRD4

As a member of the bromodomain and extraterminal domain (BET) family, BRD4 is considered as a potential target for cancer treatment. However, because of the highly conservation of its two homologous bromodomains (BD1/BD2), selective inhibition of each bromodomain remains a challenge. MS402 is a domain-selective inhibitor of BRD4-BD1 over BRD4-BD2 reported recently. Understanding the selectivity mechanism would be very useful for the further design of more potent BD1-selectivity inhibitors. Molecular dynamics simulation, adaptive biasing force and multiple-walker adaptive biasing force were performed to study the inhibition and domain-selective mechanism of MS402 toward BRD4-BD1 over BRD4-BD2 here. Results demonstrate BRD4-BD1 binds to MS402 with lower binding free energy than BRD4-BD2. Residues Gln85, Pro86, Asn140, and Ile146 are crucial for MS402's selectively binding to BRD4-BD1. MS402 needs to overcome more energy barrier to dissociate from BD1 than from BD2 pocket. These findings will be helpful for rational structural modification of existing inhibitors to increase their BD1-selectivity.     

Enantiospecificity of Chloroperoxidase-Catalyzed Epoxidation: Biased Molecular Dynamics Study of a Cis-β-Methylstyrene/Chloroperoxidase-Compound I Complex

Molecular dynamics simulations of an explicitly solvated cis-β-methylstyrene/chloroperoxidase-Compound I complex are performed to determine the cause of the high enantiospecificity of epoxidation. From the simulations, a two-dimensional free energy potential is calculated to distinguish binding potential wells from which reaction to 1S2R and 1R2S epoxide products may occur. Convergence of the free energy potential is accelerated with an adaptive biasing potential. Analysis of binding is followed by analysis of 1S2R and 1R2S reaction precursor structures in which the substrate, having left the binding wells, places its reactive double bond in steric proximity to the oxyferryl heme center. Structural analysis of binding and reaction precursor conformations is presented. We find that 1), a distortion of Glu¹⁸³ is important for CPO-catalyzed epoxidation as was postulated previously based on experimental results; 2), the free energy of binding does not provide significant differentiation between structures leading to the respective epoxide enantiomers; and 3), CPO's enantiospecificity toward cis-β-methylstyrene is likely to be caused by a specific group of residues which form a hydrophobic core surrounding the oxyferryl heme center.     

Investigation on the binding mechanism of loratinib with the c-ros oncogene 1 (ROS1) receptor tyrosine kinase via molecular dynamics simulation and binding free energy calculations

The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild–type (WT) ROS1 and ROS1^G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1^WT and apo-ROS1^G2032R systems showed similar RMSF distributions, while ROS1^G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1^G2032R significantly interfered the ?uctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design.     

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.     

Solution structure of a novel calcium binding protein, MTH1880, from Methanobacterium thermoautotrophicum

MTH1880 is a hypothetical protein from Methanobacterium thermoautotrophicum, a target organism of structural genomics. The solution structure determined by NMR spectroscopy demonstrates a typical alpha + beta-fold found in many proteins with different functions. The molecular surface of the protein reveals a small, highly acidic pocket comprising loop B (Asp36, Asp37, Asp38), the end of beta2 (Glu39), and loop D (Ser57, Ser58, Ser61), indicating that the protein would have a possible cation binding site. The NMR resonances of several amino acids within the acidic binding pocket in MTH1880, shifted upon addition of calcium ion. This calcium binding motif and overall topology of MTH1880 differ from those of other calcium binding proteins. MTH1880 did not show a calcium-induced conformational change typical of calcium sensor proteins. Therefore, we propose that the MTH1880 protein contains a novel motif for calcium-specific binding, and may function as a calcium buffering protein.     

Some insights into the binding mechanism of the GABAA receptor: a combined docking and MM-GBSA study

Gamma-aminobutyric type A receptor (GABA_AR) is a member of the Cys-loop family of pentameric ligand gated ion channels (pLGICs). It has been identified as a key target for many clinical drugs. In the present study, we construct the structure of human 2α₁2β₂γ₂ GABA_AR using a homology modeling method. The structures of ten benzodiazepine type drugs and two non-benzodiazepine type drugs were then docked into the potential benzodiazepine binding site on the GABA_AR. By analyzing the docking results, the critical residues His102 (α₁), Phe77 (γ₂) and Phe100 (α₁) were identified in the binding site. To gain insight into the binding affinity, molecular dynamics (MD) simulations were performed for all the receptor–ligand complexes. We also examined single mutant GABA_AR (His102A) in complexes with the three drugs (flurazepam, eszopiclone and zolpidem) to elucidate receptor–ligand interactions. For each receptor–ligand complex (with flurazepam, eszopiclone and zolpidem), we calculated the average distance between the C_α of the mutant residue His102A (α₁) to the center of mass of the ligands. The results reveal that the distance between the C_α of the mutant residue His102A (α₁) to the center of flurazepam is larger than that between His102 (α₁) to flurazepam in the WT type complex. Molecular mechanic-generalized Born surface area (MM-GBSA)-based binding free energy calculations were performed. The binding free energy was decomposed into ligand-residue pairs to create a ligand-residue interaction spectrum. The predicted binding free energies correlated well (R ²?=?0.87) with the experimental binding free energies. Overall, the major interaction comes from a few groups around His102 (α₁), Phe77 (γ₂) and Phe100 (α₁). These groups of interaction consist of at least of 12 residues in total with a binding energy of more than 1 kcal mol^?1. The simulation study disclosed herein provides a meaningful insight into GABA_AR–ligand interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

Impact of BRCA1 BRCT domain missense substitutions on phosphopeptide recognition

The BRCA1 BRCT domain binds pSer-x-x-Phe motifs in partner proteins to regulate the cellular response to DNA damage. Approximately 120 distinct missense variants have been identified in the BRCA1 BRCT through breast cancer screening, and several of these have been linked to an increased cancer risk. Here we probe the structures and peptide-binding activities of variants that affect the BRCA1 BRCT phosphopeptide-binding groove. The results obtained from the G1656D and T1700A variants illustrate the role of Ser1655 in pSer recognition. Mutations at Arg1699 (R1699W and R1699Q) significantly reduce peptide binding through loss of contacts to the main chain of the Phe(+3) residue and, in the case of R1699W, to a destabilization of the BRCT fold. The R1835P and E1836K variants do not dramatically reduce peptide binding, in spite of the fact that these mutations significantly alter the structure of the walls of the Phe(+3) pocket.     

Interaction of yeast tRNA(Phe) anticodon arm with 40S ribosomal subunit of rabbit liver.

The 15-nucleotide analog of yeast tRNA(Phe) anticodon arm binds cooperatively to two sites of poly(U) programmed 40S ribosome like intact tRNA(Phe). The cooperativity coefficients appeared to be about 4 for tRNA(Phe) and 50 for its anticodon arm. Anticodon arm contributes the majority of free energy of tRNA binding to a programmed 40S ribosomal subunit. The correct codon-anticodon pairing seems to play the key role in the cooperativity origin. Contrary to the anticodon arm template independent binding of the whole tRNA to the small ribosomal subunit is revealed.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

P-loop Conformation Governed Crizotinib Resistance in G2032R-Mutated ROS1 Tyrosine Kinase: Clues from Free Energy Landscape

Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance.     

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.     

Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas.     

Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.     

Interaction of human and Escherichia coli tRNA(Phe) with human 80S ribosomes in the presence of oligo- and polyuridylate templates.

Human placenta and Escherichia coli Phe-tRNA(Phe) and N-AcPhe-tRNA(Phe) binding to human placenta 80S ribosomes was studied at 13 mM Mg2+ and 20 degrees C in the presence of poly(U), (pU)6 or without a template. Binding properties of both tRNA species were studied. Poly(U)-programmed 80S ribosomes were able to bind charged tRNA at A and P sites simultaneously under saturating conditions resulting in effective dipeptide formation in the case of Phe-tRNA(Phe). Affinities of both forms of tRNA(Phe) to the P site were similar (about 1 x 10(7) M-1) and exceeded those to the A site. Affinity of the deacylated tRNA(Phe) to the P site was much higher (association constant > 10(10) M-1). Binding at the E site (introduced into the 80S ribosome by its 60S subunit) was specific for deacylated tRNA(Phe). The association constant of this tRNA to the E site when A and P sites were preoccupied with N-AcPhe-tRNA(Phe) was estimated as (1.7 +/- 0.1) x 10(6) M-1. In the presence of (pU)6, charged tRNA(Phe) bound loosely at the A and P sites, and the transpeptidation level exceeded the binding level due to the exchange with free tRNA from solution. Affinities of aminoacyl-tRNA to the A and P sites in the presence of (pU)6 seem to be the same and much lower than those in the case of poly(U). Without a messenger, binding of the charged tRNA(Phe) to 80S ribosomes was undetectable, although an effective transpeptidation was observed suggesting a very labile binding of the tRNA simultaneously at the A and P sites.     

Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.     

Phe310 in transmembrane VI of the alpha1B-adrenergic receptor is a key switch residue involved in activation and catecholamine ring aromatic bonding.

Pharmacophore mapping of adrenergic receptors indicates that the phenyl ring of catecholamine agonists is involved in receptor binding and activation. Here we evaluated Phe310, Phe311, and Phe303 in transmembrane VI (TMVI), as well as Tyr348 in TMVII of the alpha1B-adrenergic receptor (alpha1B-AR), which have been implicated in a catechol-ring interaction. Neither catecholamine docking studies nor mutagenesis studies of Phe311, Phe303, or Tyr348 supported a role for these residues in catechol-ring binding. By contrast, docking studies indicated that the Phe310 side chain is well positioned to interact with the catechol-ring, and substituted cysteine accessibility method studies revealed that the side chain of the 310, but not 311 residue, is both solvent accessible and directed into the agonist-binding pocket. Also, saturation mutagenesis of both Phe310 and Phe311 revealed for the former, but not for the latter, a direct relationship between side chain volume and agonist affinity, and that aromaticity is essential for wild-type agonist binding, and for both wild-type agonist potency and efficacy. Moreover, studies of Phe310 mutants combined with a previously described constitutively active alpha1B-AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R', interaction of Phe310 with catecholamine agonists is essential for isomerization from R' to the fully activated state, R.     

Antigen-antibody interactions and structural flexibility of a femtomolar-affinity antibody

The femtomolar-affinity mutant antibody (4M5.3) generated by directed evolution is interesting because of the potential of antibody engineering. In this study, the mutant and its wild type (4-4-20) were compared in terms of antigen-antibody interactions and structural flexibility to elucidate the effects of directed evolution. For this purpose, multiple steered molecular dynamics (SMD) simulations were performed. The pulling forces of SMD simulations elucidated the regions that form strong attractive interactions in the binding pocket. Structural analysis in these regions showed two important mutations for improving attractive interactions. First, mutation of Tyr102(H) to Ser (sequence numbering of Protein Data Bank entry 1FLR ) played a role in resolving the steric hindrance on the pathway of the antigen in the binding pocket. Second, mutation of Asp31(H) to His played a role in resolving electrostatic repulsion. Potentials of mean force (PMFs) of both the wild type and the mutant showed landscapes that do not include obvious intermediate states and go directly to the bound state. These landscapes were regarded as funnel-like binding free energy landscapes. Furthermore, the structural flexibility based on the fluctuations of the positions of atoms was analyzed. It was shown that the fluctuations in the positions of the antigen and residues in contact with antigen tend to be smaller in the mutant than in the wild type. This result suggested that structural flexibility decreases as affinity is improved by directed evolution. This suggestion is similar to the relationship between affinity and flexibility for in vivo affinity maturation, which was suggested by Romesberg and co-workers [Jimenez, R., et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100, 92-97]. Consequently, the relationship was found to be applicable up to femotomolar affinity levels.     

NMR studies of 4-19F-phenylalanine-labeled intestinal fatty acid binding protein: evidence for conformational heterogeneity in the native state

(19)F-Nuclear magnetic resonance (NMR) studies have been carried out after incorporation of 4-(19)F-phenylalanine into the intestinal fatty acid binding protein (IFABP), a protein composed of two beta-sheets containing a large hydrophobic cavity into which ligands bind. NMR spectra have been obtained with both the ligand-free and ligand-bound (oleate) forms. There are 29 residues involved in van der Waals or hydrophobic interactions or both to form a U-shaped ligand binding pocket (Sacchettni J. C., Scapin G., Gopaul D., and Gordon J. I. (1992) J. Biol. Chem. 267, 23534-23545). The protein contains eight phenylalanines, and all are included in those residues that line the pocket. Peak assignments were made using site-specific incorporation of 4-(19)F-phenylalanine. Fluorine is a highly sensitive probe to monitor the conformation and dynamics of the side chains in native state. We find that chemical exchange in the binding pocket exists in the native apo- and holo-state. Of the eight phenylalanine residues, Phe2, Phe47, Phe62, Phe68, and Phe93 are arranged on one side of the binding pocket, and all exist in two conformations with Phe2, Phe47, and Phe62 showing exchange cross-peaks with minor conformation in (19)F-(19)F nuclear Overhauser effect (NOESY) spectra. The line widths of Phe68 and Phe93 are broader than those of other phenylalanine residues and can be deconvoluted into two peaks. Phe47, Phe62, Phe68, Phe93, and Trp82 have been proposed to be involved in the early stage of collapse (Ropson, I. J., and Frieden, C. (1992) Proc. Natl. Acad. Sci U.S.A. 89, 7222-7226), but a temperature study suggests that Phe47 behaves differently than other residues and may be more involved in a later stage of folding, for example, side chain stabilization. In the holo-form, Phe17 shows an extra exchange cross-peak in addition to those exchange cross-peaks observed in apo-form. Holo-IFABP exhibits broader line width than the apo-form, suggesting more flexibility of the binding cavity upon ligand binding.