Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs.     

The wide binding properties of a wheat nonspecific lipid transfer protein. Solution structure of a complex with prostaglandin B2.

The 3D solution structure of wheat nonspecific lipid transfer protein (ns-LTP) complexed with prostaglandin B2, a lipid with both vinyl and hydroxylated groups, has been determined by 1H 2D NMR. The global fold of the protein is close to the previously published structures of wheat, maize, barley and rice ns-LTPs. The ligand is almost completely embedded in the hydrophobic core of the protein. Structure comparisons of free and bound wheat ns-LTP reveal that the binding of prostaglandin B2 hardly affects the global fold of the protein. The structural data on this unusual complex are discussed and compared with other known ns-LTP lipid-complexes.     

Solution structure of a methionine-rich 2S albumin from sunflower seeds: relationship to its allergenic and emulsifying properties

The three-dimensional structure in aqueous solution of SFA-8, a 2S albumin 103-residue protein from seeds of sunflower (Helianthus anuus L.), has been determined by NMR methods. An almost complete (1)H resonance assignment was accomplished from analysis of two-dimensional (2D) COSY and 2D TOCSY spectra, and the structure was computed by using restrained molecular dynamics on the basis of 1393 upper limit distance constraints derived from NOE cross-correlation intensities measured in 2D NOESY spectra. In contrast with most other 2S albumins, SFA-8 consists of a single polypeptide chain without any cleavage in the segment of residues 30-46. The computed structures exhibited an rmsd radius of 0.52 A for the backbone structural core (residues 11-30 and 46-101) and 1.01 A for the side chain heavy atoms. The resulting structure consists of five amphipathic helices arranged in a right-handed superhelix, a folding motif first observed in nonspecific lipid transfer (nsLTP) proteins, and common to other 2S albumins. In contrast to nsLTP proteins, neither SFA-8 nor RicC3 (a 2S albumin from castor bean) has an internal cavity that is able to host a lipid molecule, which results from an exchange in the pairing of disulfide bridges in the CXC segment. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamin superfamily, which includes a number of important food allergens. Differences in the extension and solvent exposition of the so-called "hypervariable loop" (which connects helices III and IV) in SFA-8 and RicC3 may be responsible for the different allergenic properties of the two proteins. SFA-8 has been shown to form highly stable emulsions with oil/water mixtures. We propose that these properties may be determined partly by a hydrophobic patch at the surface of the protein which consists of five methionines that partially hide the Trp76 residue. The flexibility of the loop which contains Trp76 and the hydrophobicity of the whole environment may favor a conformational change, by which the Trp76 side chain may become inserted into the oil phase.     

Complete amino acid sequence determination of the major allergen of peach (Prunus persica) Pru p 1

The major protein allergen of peach (Prunus persica), Pru p 1, has recently been identified as a lipid transfer protein (LTP). The complete primary structure of Pru p 1, obtained by direct amino acid sequence and liquid chromatography-mass spectrometry (LC-MS) analyses with the purified protein, is described here. The protein consists of 91 amino acids with a calculated molecular mass of 9178 Da. The amino acid sequence contains eight strictly conserved cysteines, as do all known LTPs, but secondary structure predictions failed to classify the peach 9 kDa protein as an 'all-alpha type', due to the high frequency of amino acids (nine prolines) disrupting alpha helices. Although the sequence similarity with maize LTP is only 63%, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP 16 are identical and 7 similar in the peach homolog, supporting the hypothesis of a similar function.     

Conformational plasticity of the lipid transfer protein SCP2

The nonspecific lipid transfer protein sterol carrier protein 2 (SCP2) is involved in organellar fatty acid metabolism. A hydrophobic cavity in the structure of SCP2 accommodates a wide variety of apolar ligands such as cholesterol derivatives or fatty acyl-coenzyme A (CoA) conjugates. The properties of this nonspecific lipid binding pocket are explored using NMR chemical shift perturbations, paramagnetic relaxation enhancement, amide hydrogen exchange, and 15N relaxation measurements. A common binding cavity shared by different physiological ligands is identified. NMR relaxation measurements reveal that residues in the three C-terminal alpha-helices within the lipid binding region exhibit mobility at fast (picosecond to nanosecond) and slow (microsecond to millisecond) time scales. Ligand binding is associated with a considerable loss of peptide backbone mobility. The observed conformational dynamics in SCP2 may play a role for the access of hydrophobic ligands to an occluded binding pocket. The C-terminal peroxisomal targeting signal of SCP2 is specifically recognized by the Pex5p receptor protein, which conducts cargo proteins toward the peroxisomal organelle. Neither the C-terminal targeting signal nor the N-terminal precursor sequence interferes with lipid binding by SCP2. The alpha-helices involved in lipid binding also mediate a secondary interaction interface with the Pex5p receptor. Silencing of conformational dynamics of the peptide backbone in these helices upon either lipid or Pex5p binding might communicate the loading state of the cargo protein to the targeting receptor.     

Solution structure of chicken liver basic fatty acid binding protein

Chicken liver basic fatty acid binding protein (Lb-FABP) belongs to the basic-type fatty acid binding proteins, a novel group of proteins isolated from liver of different non mammalian species whose structure is not known. The structure of Lb-FABP has been solved by ¹H NMR. The overall fold of Lb-FABP, common to the other proteins of the family, consists of ten antiparallel -strands organised in two nearly ortogonal -sheets with two alpha helices closing the protein cavity where small hydrophobic ligands can be bound. The binding specificity of the protein is not known, however, based on the high sequence and structural similarity with an orthologous protein, ileal lipid binding protein, it is suggested that bile acids may be the putative ligands.     

Protein stability and plasticity of the hydrophobic cavity in wheat ns-LTP

Plant ns-LTPs display an original structure with four helices and a flexible C-terminus, maintained together by four disulphide bridges and delineating an elongated central hydrophobic cavity. In order to relate these structural features to the protein stability and plasticity, combined molecular mechanics and simulated annealing calculations were undertaken on a wheat ns-LTP "mutant" with Cys-Ala replacement and with the application of core inter-residue restraints up to 2 A, reducing the cross-section size of the hydrophobic cavity. Analysis of the energy-minimized structures shows that removal of the disulphide bridges results in structures with a lower total energy and a smaller cavity volume. A 1-ns MD simulation at 300K in water, underlines that, despite the absence of a well-packed hydrophobic core, the native structure is extremely stable at room temperature and the cavity is not hydrated. This confirms that the disulphide bridges are essential for the existence of the cavity, whereas its plasticity depends both on the hydrophobic chain lining the cavity and on the C-terminal flexibility. A high temperature (500K) MD simulation confirms the stability of the secondary structure elements and the flexibility of the loops and of the C-terminal segment. Two important structural transitions during this simulation are discussed and possible routes for the insertion and release of hydrophobic ligands are suggested.     

Identification of two lipid binding proteins from liver of Gallus domesticus

1. Two low molecular weight (approximately 14,000 Da) proteins exhibiting lipid binding activity were purified from liver cytosol and identified as non-specific lipid binding protein (ns-LTP) and fatty acid binding protein (L-FABP). 2. Ligand binding assays indicated that ns-LTP exhibited greater binding activity for cholesterol and little binding of fatty acids. Conversely, L-FABP had higher relative binding activity for fatty acids but did not bind cholesterol. 3. Amino acid composition and pI data supported the identification of the chicken liver lipid binding proteins as L-FABP and ns-LTP. 4. Polyclonal antisera was prepared against each of the liver lipid binding proteins and monospecificity verified using Western blot analysis.     

Structure of a specialized acyl carrier protein essential for lipid a biosynthesis with very long-chain Fatty acids in open and closed conformations

The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 ? resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.     

Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens

This study describes the three-dimensional crystal structure of a non-specific lipid transport protein (ns-LTP) from Rosaceae. Whilst ns-LTPs from species other than Rosaceae, such as nuts, cereals, grape, oranges and vegetables are also responsible for plant food allergies, this is less frequent compared with ns-LTPs from Rosaceae in the Mediterranean area. In this heterologously expressed peach Pru p3, a ligand is present inside the central cavity of the protein, presumably a fatty acid that was present or produced in the culture medium of the expression organism Escherichia coli. Moreover, the two molecules of ns-LTP present in the asymmetric unit bind this ligand in a different way, suggesting a significant degree of plasticity for the peach ns-LTP binding cavity, despite the presence of four disulphide bridges. Two molecules are present in the asymmetric unit: molecule A is a fully liganded protein, while molecule B apparently represents a partially liganded state. Also, molecular dynamics simulation, along with other evidence, suggests that these two molecular conformations represent different states in solution. Comparison of the 3D models of different ns-LTPs justifies the evidence of a high degree of conservation of the putative IgE binding epitopes among proteins of the Rosaceae family and the presence of significant amino acid replacements in correspondence of the same regions in ns-LTPs of botanical species unrelated to Rosaceae.     

A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins

Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.     

Fluorine-19 NMR studies on the acid state of the intestinal fatty acid binding protein

The intestinal fatty acid binding protein (IFABP) is composed of two beta-sheets with a large hydrophobic cavity into which ligands bind. After eight 4-(19)F-phenylalanines were incorporated into the protein, the acid state of both apo- and holo-IFABP (at pH 2.8 and 2.3) was characterized by means of (1)H NMR diffusion measurements, circular dichroism, and (19)F NMR. Diffusion measurements show a moderately increased hydrodynamic radius while near- and far-UV CD measurements suggest that the acid state has substantial secondary structure as well as persistent tertiary interactions. At pH 2.8, these tertiary interactions have been further characterized by (19)F NMR and show an NOE cross-peak between residues that are located on different beta-strands. Side chain conformational heterogeneity on the millisecond time scale was captured by phase-sensitive (19)F-(19)F NOESY. At pH 2.3, native NMR peaks are mostly gone, but the protein can still bind fatty acid to form the holoprotein. An exchange cross-peak of one phenylalanine in the holoprotein is attributed to increased motional freedom of the fatty acid backbone caused by the slight opening of the binding pocket at pH 2.8. In the acid environment Phe128 and Phe17 show dramatic line broadening and chemical shift changes, reflecting greater degrees of motion around these residues. We propose that there is a separation of specific regions of the protein that gives rise to the larger radius of hydration. Temperature and urea unfolding studies indicate that persistent hydrophobic clusters are nativelike and may account for the ability of ligand to bind and induce nativelike structure, even at pH 2.3.     

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds.

The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).     

Structure of sterol carrier protein 2 at 1.8 A resolution reveals a hydrophobic tunnel suitable for lipid binding

Sterol carrier protein 2, also known as nonspecific lipid transfer protein is a ubiquitous, small, basic protein of 13 kDa found in animals. Its primary structure is highly conserved between different species, and it has been implicated in the intracellular transport of lipids and in a wide range of other in vitro functions related to sterol and fatty acid metabolism. Sterol carrier protein 2 deficiency in mice leads to elevated concentrations of phytanic acid in the serum and causes hepatocarcinogenesis. However, its actual physiological role is still unknown. Although sterol carrier protein 2 has been studied extensively in the past 20 years, very little is known concerning its three-dimensional structure. The crystal structure of rabbit sterol carrier protein 2, determined at 1.8 A resolution with the MIRAS method, shows a unique alpha/beta-fold. The core of this protein forms a five-stranded antiparallel beta-sheet flanked by five helices. A C-terminal segment (residues 114-123), together with part of the beta-sheet and four alpha-helices, form a hydrophobic tunnel providing the environment for apolar ligands such as fatty acids and fatty acyl-coenzyme As. Structurally well-characterized nonspecific lipid transfer proteins from plants have hydrophobic tunnel-like cavities, which were identified as the binding site for fatty acids and related apolar ligands. Despite the fact that plant nonspecific lipid transfer proteins are smaller proteins than sterol carrier protein 2, show no sequence homology to sterol carrier protein 2, and are structurally unrelated, the cavities of these two classes of proteins are very similar with respect to size, shape, and hydrophobicity, suggesting a common functional role.     

A novel lipid transfer protein from the dill Anethum graveolens L.: isolation,structure, heterologous expression,and functional characteristics

A novel lipid transfer protein, designated as Ag‐LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag‐LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag‐LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag‐LTP spatial structure contains four α ‐helices, forming the internal hydrophobic cavity, and a long C‐terminal tail. The measured volume of the Ag‐LTP hydrophobic cavity is equal to ~800 A³, which is much larger than those of other plant LTP1s. Ag‐LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Magnetization transfer from laser-polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer protein

Nonspecific lipid transfer protein from wheat is studied by liquid-state NMR in the presence of xenon. The gas-protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser-polarized xenon to the protein protons. Twenty-six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns-LTP cavity. Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by approximately 12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.     

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.     

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.