NMR investigations of protein-carbohydrate interactions binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N', N"-triacetylchitotriose.

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1H-NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative DeltaH which is partially compensated by negative DeltaS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 A, while the heavy atom rmsd was 2.10 A. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three-dimensional structure of WGA-B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.     

NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.     

Solution structure of a steroid-DNA complex with cholic acid residues sealing the termini of a Watson-Crick duplex

The three-dimensional structure of a covalent hybrid between cholic acid and the self-complementary DNA hexamer 5'-TGCGCA-3' was solved via two-dimensional NMR and restrained torsion angle molecular dynamics. In the complex, refined to a pairwise rmsd of 0.64 A, the steroid binds to the terminal T:A base pairs via extensive van der Waals contacts but without any hydrogen bonds or detectable dipole-dipole interactions. The contacts involve the methyl groups as well as one edge of the streoid's sterane skeleton and both nucleobases and the deoxyriboses of the terminal base pair of the DNA. The surprising shape complementarity between steroid and the undisturbed DNA termini explains the increase in fidelity and affinity observed for hybridization probes bearing bile acid residues. Since the hydroxyl groups of the steroid do not contribute to the binding of the DNA, they may be derivatized, potentially giving access to a new class of specific binders for blunt ends of Watson-Crick duplexes.     

Three-dimensional structures of complexes of Lathyrus ochrus isolectin I with glucose and mannose: fine specificity of the monosaccharide-binding site

The structure of the methyl-alpha-D-mannopyranoside-LOL I complex has been solved by the molecular replacement method using the refined saccharide-free LOL I coordinates as starting model. The methyl-alpha-D-mannopyranoside-LOL I complex was refined by simulated annealing using the program X-PLOR. The final R-factor value is 0.182 [Fo greater than 1 sigma(Fo)]. The isostructural methyl-alpha-D-glucopyranoside-LOL I complex was refined by X-Ray coupled energy minimization using the methyl-alpha-D-mannopyranoside-LOL I structure as a starting model to an R factor of 0.179 (all data). In both crystal forms, each dimer binds two molecules of sugar in pockets found near the calcium ions. The two saccharide moieties, which are in the C1 chair conformation, establish the same hydrogen bond pattern with the lectin. However, the van der Waals contacts are different between the O2, C2, C6, and O6 atoms of the two molecules and the backbone atoms of residues 208-211. Mannose, due to its axial C2 conformation, encloses the backbone atoms of the protein in a clamplike way. Van der Waals energy calculations suggest that this better complementarity of the mannoside molecule with the lectin could explain its higher affinity for isolectin I.     

Molecular interaction of manganese based carbon monoxide releasing molecule (MnCORM) with human serum albumin (HSA)

In the present study, the interaction between the HSA and MnCORM in vitro under physiological conditions, was investigated through ultraviolet-visible (UV-vis) absorption, fluorescence, time-resolved fluorescence, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic techniques and in silico molecular docking methods. Binding parameters such as the binding constant, number of binding sites and binding force were obtained from the fluorescence data. Thermodynamic interaction revealed that the reaction was spontaneous (ΔG < 0) and hydrogen bond and van der Waals interaction were primarily involved in the binding. The changes induced in the secondary structure conformation due to the MnCORM interaction were monitored using CD and FT-IR spectroscopic techniques. The results showed reduction in α-helix conformation and corresponding increase in β-sheet and unordered structures due to slight unfolding. The time-resolved fluorescence decay confirmed the static quenching mechanism of the MnCORM. The molecular docking studies revealed that the MnCORM interacted at Sudlow’s site II of domain IIIA through hydrogen bond and van der Waals interactions. In order to understand the drug distribution and elimination, studies on the drug molecule interaction with HSA are vital. Therefore, it is evident that MnCORM interacts with HSA through ground state complex formation and thus suitable for in vivo delivery.     

Insights into the binding of ferulic acid to the thermally treated xanthine oxidase

Ferulic acid (FA) is a biologically active compound used as an additive in the food industry, and possesses a wide range of therapeutic effects for treating different health problems. The interaction between FA and bovine xanthine oxidase (XOD) has been investigated by means of fluorescence spectroscopy methods. The numbers of binding sites and the binding constants were estimated at various temperatures and the results indicated the existence of one specific FA binding site of XOD. Detailed information on the interaction between molecules gathered after performing in silico molecular docking indicated the accommodation of the FA molecule in a XOD binding pocket, in close vicinity to the active site residues. The formation of the XOD–FA complex causes the quenching of protein fluorescence. The process followed a static mechanism at lower temperatures, and a dynamic mechanism at higher temperatures. The thermodynamic parameters calculated on the basis of different temperatures revealed that the association between FA and XOD is a spontaneous process driven by enthalpy and dominated by hydrogen bonding and van der Waals interaction. The results of synchronous fluorescence and 3D fluorescence spectra showed that the conformation of protein was altered in the presence of FA. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular dynamics studies of the HIV-1 TAR and its complex with argininamide

The dynamic behavior of HIV-1 TAR and its complex with argininamide is investigated by means of molecular dynamics simulations starting from NMR structures, with explicit inclusion of water and periodic boundary conditions particle mesh Ewald representation of the electrostatic energy. During simulations of free and argininamide-bound TAR, local structural patterns, as determined by NMR experiments, were reproduced. An interdomain motion was observed in the simulations of free TAR, which is absent in the case of bound TAR, leading to the conclusion that the free conformation of TAR is intrinsically more flexible than the bound conformation. In particular, in the bound conformation the TAR–argininamide interface is very well ordered, as a result of the formation of a U·A·U base triple, which imposes structural constraints on the global conformation of the molecule. Free energy analysis, which includes solvation contributions, was used to evaluate the influence of van der Waals and electrostatic terms on formation of the complex and on the conformational rearrangement from free to bound TAR.     

The phage 434 Cro/OR1 complex at 2.5 A resolution

The crystal structure of phage 434 Cro protein in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution. The DNA fragment contains the sequence of the OR1 operator site. The structure shows a bent conformation for the DNA, straighter at the center and more bent at the ends. The central base-pairs adopt conformations with significant deviations from coplanarity. The two molecules interact extensively along their common interface, both through hydrogen bonds and van der Waals interactions. The significance of these interactions for operator binding and recognition is discussed.     

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

The assignment of the ¹H and ¹⁵Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.     

The impact of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Cel7A carbohydrate binding domain mutations on its binding to a cellulose surface: a molecular dynamics free energy study

A critical role of the Family 7 cellobiohydrolase (Cel7A) carbohydrate binding domain (CBD) is to bind to a cellulose surface and increase the enzyme concentration on the surface. Several residues of Trichoderma reesei Cel7A CBD, including Y5, N29, Y31, Y32 and Q34, contribute to cellulose binding, as revealed by early experimental studies. To investigate the interactions between these important residues and cellulose, we applied a thermodynamic integration method to calculate the cellulose–Cel7A CBD binding free energy changes caused by Y5A, N29A, Y31A, Y32A and Q34A mutations. The experimental binding trend was successfully predicted, proving the effectiveness of the complex model. For the two polar residue mutants N29A and Q34A, the changes in the electrostatics are comparable to those of van der Waals, while for three Y to A mutants, the free energy differences mainly come from van der Waals interactions. However, in both cases, the electrostatics dominates the interactions between individual residues and cellulose. The side chains of these residues are rigidified after the complex is formed. The binding free energy changes for the two mutants Y5W and Y31W were also determined, and for these the van der Waals interaction was strengthened but the electrostatics was weakened.     

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.     

Direct interactions in the recognition between the environmental estrogen bisphenol AF and human serum albumin

Bisphenol AF (BPAF) was used as a model compound to investigate the binding mechanism between the endocrine disrupting compound and human serum albumin (HSA) using multispectroscopic techniques and molecular modeling method at the protein level. The results indicated that BPAF was indeed bound to HSA and located in the hydrophobic pocket of HSA on subdomain IIA through hydrogen bond and van der Waals interactions. The fluorescence quenching data showed that the binding of BPAF and HSA quenched the intrinsic fluorescence of HSA, and the static quenching constants were acquired. Copyright © 2015 John Wiley & Sons, Ltd.     

Solution structure of the 40,000 Mr phosphoryl transfer complex between the N-terminal domain of enzyme I and HPr

The solution structure of the first protein-protein complex of the bacterial phosphoenolpyruvate: sugar phosphotransferase system between the N-terminal domain of enzyme I (EIN) and the histidine-containing phosphocarrier protein HPr has been determined by NMR spectroscopy, including the use of residual dipolar couplings that provide long-range structural information. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2', 3 and 4 of the alpha-subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer.     

Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions

The structure ofE. coli-derived rat intestinal fatty acid-binding protein has recently been refined to 1.2 Å without bound fatty acid and to 2.0 Å and 1.75 Å with bound hexadecanoate (palmitate) and 9Z-octadecenoate (oleate), respectively. The structure ofE. coli-derived human muscle fatty acid-binding protein has also been solved to 2.1 Å with a C16 bacterial fatty acid. Both proteins contain 10 anti-parallel -strands in a+1, +1, +1... motif. The strands are arranged in two -pleated sheets that are orthogonally oriented. In each case, the fatty acid is enclosed by the -sheets and is bound to the proteins by feeble forces. These feeble forces consist of (i) a hydrogen bonding network between the fatty acid's carboxylate group, ordered solvent, and side chains of polar/ionizable amino acid residues; (ii) van der Waals contacts between the methylene chain of the fatty acid and the side chain atoms of hydrophobic and aromatic residues; (iii) van der Waals interactions between the methyl and the component methenyls of the phenyl side chain of a Phe which serves as an adjustable terminal sensor situated over a surface opening or portal connecting interior and exterior solvent; and (iv) van der Waals contacts between methylenes of the alkyl chain and oxygens of ordered waters that have been located inside the binding cavity. These waters are positioned over one face of the ligand and are held in place by hydrogen bonding with one another and with the side chains of protein's polar and ionizable residues. Binding of the fatty acid ligand is associated with minimal adjustments of the positions of main chain or side chain atoms. However, acquisition of ligand is associated with removal of ordered interior solvent suggesting that the free energy of dehydration of the binding site may be as important for the energy of the binding reaction as the free energy of stabilization of the fatty acid: protein complex.     

Molecular basis for triclosan activity involves a flipping loop in the active site

The crystal structure of the Escherichia coli enoyl reductase-NAD+-triclosan complex has been determined at 2.5 A resolution. The Ile192-Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure-based inhibitor design.     

FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 A resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site.     

The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis

The three-dimensional structure of the maltose- or maltodextrin-binding protein (Mr = 40,622) with bound maltose has been obtained by crystallographic analysis at 2.8-A resolution. The structure, which has been partially refined at 2.3 A, is ellipsoidal with overall dimensions of 30 x 40 x 65 A and divided into two distinct globular domains by a deep groove. Although each domain is built from two peptide segments from the amino- and carboxyl-terminal halves, both domains exhibit similar supersecondary structure, consisting of a central beta-pleated sheet flanked on both sides with two or three parallel alpha-helices. The groove, which has a depth of 18 A and a base of about 9 x 18 A, contains the maltodextrin-binding site. We have previously observed the same general features in the well-refined structures of six other periplasmic receptors with specificities for L-arabinose, D-galactose/D-glucose, sulfate, phosphate, leucine/isoleucine/valine, and leucine. The bound maltose is buried in the groove and almost completely inaccessible to the bulk solvent. The groove is heavily populated by polar and aromatic groups many of which are involved in extensive hydrogen-bonding and van der Waals interactions with the maltose. All the disaccharide hydroxyl groups, which form a peripheral polar surface approximately in the plane of the sugar rings, are tied in a total of 11 direct hydrogen bonds with six charged side chains, one Trp side chain, and one peptide backbone NH, and five indirect hydrogen bonds via water molecules. The maltose is wedged between four aromatic side chains. The resulting stacking of these aromatic residues on the faces of the glucosyl units provides a majority of the van der Waals contacts in the complex. The nonreducing glucosyl unit of the maltose is involved in approximately twice as many hydrogen bonds and van der Waals contacts as the glucosyl unit at the reducing end. The binding protein-maltose complex shows the best example of the extensive use of polar and aromatic residues in binding oligosaccharides. The tertiary structure of the maltodextrin-binding protein, along with the results of genetic studies by a number of investigators, has also enabled us for the first time to map the different regions on the surface of the protein involved in the interactions with the membrane-bound protein components necessary for transport of and chemotaxis toward maltodextrins. These sites permit distinction of the "open cleft" (without bound sugar) and closed (with bound sugar) conformations of the binding protein by the chemotactic signal transducer with which the maltodextrin-binding protein interacts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel homodimer model of the β-adrenergic receptor in complex with free fatty acids and cholesterol: first-principles calculation studies

We propose a theoretical novel homodimer model of the β- adrenergic receptor (βAR) in complex with a heterogeneous mixture of free fatty acids (FFAs) and cholesterol based on first-principles calculations. We used the density-functional-based tight binding with dispersion (DFTB-D) method, which accurately evaluates van der Waals interactions between FFAs and amino acid residues in the receptor. The calculations suggest that a stable homodimer of bAR can form a complex with FFAs and cholesterol by extensive van der Waals interactions in the cell membrane, and that the heterogeneous composition of the FFAs is important for the stability of the homodimer complex. The stable van der Waals interactions propagate from one of the bAR to the other through the cholesterol and FFAs in the homodimer complex. The energy propagation in the complex has the potential to enhance molecular signaling in adipocytes, because the stability of the complex can influence anti-adiposity effects after oral treatment of the FFA components.     

Prediction of fluorine chemical shifts in proteins

Molecular dynamics calculations have been used in an effort to estimate the change in fluorine nmr shielding when a fluorine nucleus enters the tertiary structure of a protein. Considerations of the possible interactions that can define the shift parameter change suggest that van der Waals interactions are the leading determinant of fluorine shifts in proteins, although aromatic ring currents, other magnetic anisotropies, and electrostatic field effects could result in shift distinctions of 1 ppm or smaller. Results of our studies of a fluorine-containing analogue of the ribonuclease A S-protein/S-peptide complex indicate that static structures such as those implied by crystallographic data lead to overestimates of the magnitude of the van der Waals shielding term; molecular dynamics simulations provide indications of the effects of conformational averaging in defining this term. The treatment used predicts the correct direction of the shift change when the fluorine enters this protein environment from aqueous solution and, with an experimentally supported choice of adjustable parameters, gives agreement with the magnitude of the shift.