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Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics.

In circulation HSA is the principal carrier for endogenous lipophilic compounds, primarily non-esterified long chain fatty acids (FA). Since FA bind to multiple binding sites with varying affinities, it would be useful to probe the relative affinities for these FA binding sites with a method that distinguishes individual binding sites. As visualized in crystal structures FA binding distributes asymmetrically throughout the protein. In the physiologically relevant solution state, these sites were partially distinguished by 1D-13C-NMR spectroscopy and subsequently correlated with crystal structure sites. Here we show nine, well-resolved peaks in 1H-13C-NMR spectra of 18-13C-oleic acid (OA) / HSA complexes. Different NMR signals represent FA bound at different sites throughout the protein, with the varying intensities corresponding to the different affinities HSA has for OA. This investigation probes the relative affinities of FA with three approaches: (i) addition of different FA molar ratios to HSA, to observe the order of filling of binding sites; (ii) addition of FA acceptors to observe the dissociation of FA from HSA; and (iii) addition of drugs that are known to bind to low affinity FA sites. From the order of filling, the three highest affinity-binding sites are clearly differentiated from the six lower/medium affinity-binding sites at the physiologically relevant FA:HSA molar ratio- 4:1. Methyl-β-cyclodextrin (MβCD) extracted FA from individual sites, in a concentration dependent manner, with the highest concentrations removing FA from the highest affinity sites. Relative affinities determined as above were consistent with the binding of drugs to Sudlow's drug binding sites, which displaced bound FA from specific lower affinity sites. 2D-NMR spectroscopy is a powerful approach for studying interactions of FA with HSA and FA-competitive drugs in a site-specific manner, providing a unique view of HSA-ligand binding.

Storage of lipid in ectopic depots outside of abdominal visceral and subcutaneous stores, including within the pericardium and liver, has been associated with obesity, insulin resistance, and cardiovascular risk. We sought to determine whether anatomically distinct ectopic depots were physiologically correlated and site-specific effects upon cardiovascular function could be identified. Obese subjects (n = 28) with metabolic syndrome but without known atherosclerotic disease and healthy controls (n = 18) underwent magnetic resonance imaging (MRI) and proton MR spectroscopy (MRS) to quantify pericardial and periaortic lipid volumes, cardiac function, aortic compliance, and intrahepatic lipid content. Fasting plasma lipoproteins, glucose, insulin, and free-fatty acids were measured. Pericardial and intrahepatic (P < 0.01) and periaortic (P < 0.05) lipid volumes were increased in obese subjects vs. controls and were strongly and positively correlated (P ≤ 0.01) but independent of BMI (P = NS) among obese subjects. Intrahepatic lipid was associated with insulin resistance (P < 0.01) and triglycerides (P < 0.05), whereas pericardial and periaortic lipid were not (P = NS). Periaortic and pericardial lipid positively correlated to free-fatty acids (P ≤ 0.01) and negatively correlated to high-density lipoprotein (HDL) cholesterol (P < 0.05). Pericardial lipid negatively correlated to cardiac output (P = 0.03) and stroke volume (P = 0.01) but not to left ventricular ejection fraction (P = 0.46). None of the ectopic depots correlated to aortic compliance. In conclusion, ectopic storage of lipid in anatomically distinct depots appeared tightly correlated but independent of body size. Site-specific functional abnormalities were observed for pericardial but not periaortic lipid. These findings underscore the utility of MRI to assess individual differences in ectopic lipid that are not predictable from BMI.

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co- and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-R-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-‚. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substrate- binding domain, but relative affinities differ. The binding specificity of DnaK- ‚ is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-‚ upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding ¢G° of 3.1 ( 0.9 kcal mol -1 and an m value of 1.7 ( 0.4 kcal mol -1 M -1 . ‡ Joint first authors. §

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.

Polymerization of the soluble beta-amyloid peptide into highly ordered fibrils is hypothesized to be a causative event in the development of Alzheimer's disease. Understanding the interactions of Abeta with inhibitors on an atomic level is fundamental for the development of diagnostics and therapeutic approaches, and can provide, in addition, important indirect information of the amyloid fibril structure. We have shown recently that trRDCs can be measured in solution state NMR for peptide ligands binding weakly to amyloid fibrils. We present here the structures for two inhibitor peptides, LPFFD and DPFFL, and their structural models bound to fibrillar Abeta(14-23) and Abeta(1-40) based on transferred nuclear Overhauser effect (trNOE) and transferred residual dipolar coupling (trRDC) data. In a first step, the inhibitor peptide structure is calculated on the basis of trNOE data; the trRDC data are then validated on the basis of the trNOE-derived structure using the program PALES. The orientation of the peptide inhibitors with respect to Abeta fibrils is obtained from trRDC data, assuming that Abeta fibrils orient such that the fibril axis is aligned in parallel with the magnetic field. The trRDC-derived alignment tensor of the peptide ligand is then used as a restraint for molecular dynamics docking studies. We find that the structure with the lowest rmsd value is in agreement with a model in which the inhibitor peptide binds to the long side of an amyloid fibril. Especially, we detect interactions involving the hydrophobic core, residues K16 and E22/D23 of the Abeta sequence. Structural differences are observed for binding of the inhibitor peptide to Abeta14-23 and Abeta1-40 fibrils, respectively, indicating different fibril structure. We expect this approach to be useful in the rational design of amyloid ligands with improved binding characteristics.

In this communication, we suggest that transferred residual dipolar couplings (trRDCs) can be employed to restrain the structure of peptide inhibitors transiently binding to beta-amyloid fibrils. The effect is based on the spontaneous alignment of amyloid fibrils with the fibril axis parallel to the magnetic field. This alignment is transferred to the transiently binding peptide inhibitor and is reflected in the size of the trRDCs. We find that the peptide inhibitor adopts a beta-sheet conformation with the backbone N-H and C-H dipolar vectors aligned preferentially parallel and perpendicular, respectively, to the fibril axis.

Spectral resolution imposes a major problem on the evaluation of MAS solid-state NMR experiments as larger biomolecular systems are concerned. We show in this communication that decoupling of the (13)C-(13)C homonuclear scalar couplings during stroboscopic detection can be successfully applied to increase the spectral resolution up to a factor of 2-2.5 and sensitivity up to a factor of 1.2. We expect that this approach will be useful for the study of large biomolecular systems like membrane proteins and amyloidogenic peptides and proteins where spectral overlap is critical. The experiments are demonstrated on a uniformly (13)C,(15)N-labelled sample of Nac-Val-Leu-OH and applied to a uniformly (13)C,(15)N-enriched sample of a hexameric amyloidogenic peptide.

Neutron emission measurement at the HL-2A tokamak device with a liquid scintillation detector is described. The detector was placed at a location with little structure material in the field of view, and equipped with a gain monitoring system which could provide the possibility to evaluate the gain variation as well as to correct for the detector response. Time trace of the neutron emissivity was obtained and it was consistent with the result of a standard (235)U fission chamber. During the plasma discharge the neutron yield could vary by about four orders of magnitude and the fluctuation of the detector gain was up to about 6%. Pulse height spectrum of the liquid scintillation detector was constructed and corrected with the aid of the gain monitoring system, and the correction was found to be essential for the assessment of the neutron energy spectrum. This successful measurement offered experience and confidence for the application of liquid scintillation detectors in the upcoming neutron camera system.

A compact neutron spectrometer based on the liquid scintillator is presented for neutron energy spectrum measurements at the HL-2A tokamak. The spectrometer was well characterized and a fast digital pulse shape discrimination software was developed using the charge comparison method. A digitizer data acquisition system with a maximum frequency of 1 MHz can work under an environment with a high count rate at HL-2A tokamak. Specific radiation and magnetic shielding for the spectrometer were designed for the neutron spectrum measurement at the HL-2A tokamak. For pulse height spectrum analysis, dedicated numerical simulation utilizing NUBEAM combined with GENESIS was performed to obtain the neutron energy spectrum. Subsequently, the transportation process from the plasma to the detector was evaluated with Monte Carlo calculations. The distorted neutron energy spectrum was folded with the response matrix of the liquid scintillation spectrometer, and good consistency was found between the simulated and measured pulse height spectra. This neutron spectrometer based on a digital acquisition system could be well adopted for the investigation of the auxiliary heating behavior and the fast-ion related phenomenon on different tokamak devices., Comment: 17 pages,9 figures

The neutron emission spectrum from neutral beam-heated plasmas of the Experimental Advanced Superconducting Tokamak (EAST) is investigated based on first-principles simulations of the fast deuteron energy distribution. Parametrized plasma profiles are used to determine the beam deposition and fast deuteron distribution function using the NUBEAM code. The deuteron distribution is also interpreted with the help of empirical models and the components in the neutron energy spectrum are computed for different viewing lines using a Monte Carlo method. The resulting observational possibilities are discussed for the case of a time-of-flight neutron emission spectrometer optimized for EAST.

A new compact stilbene crystal neutron spectrometer has been investigated and applied in the neutron emission spectroscopy on the EAST tokamak. A new components analysis method is presented to study the anisotropic light output in the stilbene crystal detector. A Geant4 code was developed to simulate the neutron responses in the spectrometer. Based on both the optimal light output function and the fitted pulse height resolution function, a reliable neutron response matrix was obtained by Geant4 simulations and validated by 2.5 MeV and 14 MeV neutron measurements at a 4.5 MV Van de Graaff accelerator. The spectrometer was used to diagnose the ion temperature in plasma discharges with lower hybrid wave injection and ion cyclotron resonance heating on the EAST tokamak.

Liver Fatty Acid Binding Protein (L-FABP), a small (14 kDa) abundant cytosolic protein, may perform several functions in cells, including intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein (iLBP) family, L-FABP is very unique in its ability to bind two molecules of FA and a variety of other bulky ligands such as bilirubin. To help understand the promiscuous binding and transport properties of L-FABP, we have applied multi-dimensional homonuclear and heteronuclear NMR spectroscopy for studies of its structure and ligand binding. The overall conformation of human L-FABP, as determined from NOE-derived distance restraints, shows a β-clam motif comprised of a 10-stranded anti-parallel β-sheet that is covered by 2 short nearly parallel α-helices. Ligand binding to L-FABP is being studied by NMR titration experiments with two types of ligands. In the case of oleic acid, which is the primary physiological ligand of L-FABP, 2D HSQC spectra with different binding stoichiometries showed two binding sites with different affinities. In addition, two 13C-labeled bilirubin analogs are being studied to assess binding of bulky ligands. We hypothesize that the unique binding of bulky hydrophobic ligands enables the L-FABP to undergo a conformational change that is different from the other FABPs.

The most abundant protein in plasma, human serum albumin (HSA), is the principal carrier for endogenous lipophilic compounds and lipophilic drugs. Non-esterified long chain fatty acids (FA) are its primary physiological ligand, with multiple binding sites of varying affinities. Detailed structural understandings of HSA-ligand interactions are vital during the development of drugs. HSA ligand interactions have been studied by various biophysical methods: x-ray crystallography, fluorescence spectroscopy, and nuclear magnetic resonance (NMR). Numerous crystal structures of HSA, with and without drugs and/or FAs, have been published. Compared to crystallography, our solution state NMR studies are focused on the more physiologically relevant state. Previously, we identified three high affinity and several low affinity FA binding sites on HSA by 1D-NMR at low resolution. Now, we are correlating crystallographic data with high-resolution 2D-NMR spectra of 13C-methyl-labeled oleic acid (OA). Our 2D-HSQC spectra show the 13C-methyl group of OA bound to HSA. The unique microenvironments that each methyl group experiences in the binding pockets gives rise to different NMR signals of varying intensities. These correspond to the different affinities that HSA has for OA, and our 2D-NMR spectra clearly differentiate the three highest affinity-binding sites from six lower affinity-binding sites. We have identified Sudlow's Drug Sites by utilizing the known FA competitors and Sudlow's site binders: warfarin and diazepam. By expanding this drug competition strategy with ibuprofen and diflunisal, we have identified additional FA binding sites. The highest affinity FA binding sites do not have known competitors and these sites are identified with the use of site-directed mutagenesis of HSA. In summary, we are reporting an approach for studying the interactions of FAs with HSA and FA-competitive drugs in a site-specific manner through the use of state-of-the-art 2D-NMR techniques.