Halogenated natural products constitute diverse and promising feedstock for molecular pharmaceuticals. However, their solution-structure elucidation by NMR presents several challenges, including the lack of fast methods to compute C chemical shifts for carbons bearing heavy atoms. We show that parametric corrections to DFT-computed chemical shifts in conjunction with rff-computed spin-spin coupling constants allow for fast and reliable screening of a large number of reported halogenated natural products, resulting in expedient structure validation or revision. In this paper, we examine more than 100 structures of halogenated terpenoids and other natural products with the new parametric approach and demonstrate that the accuracy of the combined method is sufficient to identify misassignments and suggest revisions in most cases (16 structures are revised). As the 1DH and C NMR data are ubiquitous and most routinely used in solution structure elucidation, this fast and efficient two-criterion method (nuclear spin-spin coupling andC chemical shifts) which we term DU8+ is recommended as the first essential step in structure assignment and validation.

The validity of the popular point dipole approximation for interpretation of the zero-field splitting (ZFS) parameter (D-value) in EPR spectroscopy is studied. This approximation is of central importance for the determination of distances by analysis of EPR data. In this work, a detailed experimental (EPR spectroscopy and X-ray crystallography) and theoretical study for a model system (2,2’,5,5’-tetra(t-butyl)-4,4’-bis(ethoxy-carbonyl)-3,3’-bipyrrolyl-1,1’-dioxyl) was performed to understand the scope and limitations of the point dipole model in EPR spectroscopy. For this diradical, the radical-radical distance derived with the point-dipole approximation deviates significantly (by ~40%) from the results derived from the X-ray analysis. Explicit quantum chemical calculation of the D-value on the basis of B3LYP density functional calculations leads to excellent quantitative agreement with the measured D-value. The quantitative accuracy of the employed methodology was confirmed for two additional systems that have previously been experimentally characterized. We therefore analyzed the contributions to the D-value of the target system in detail. This analysis leads to insight into the reasons for the failure of the point-dipole approximation. The analysis was then extended to an in silico study of five classes of model systems. Linkers of varying length and bond saturation were introduced between the radical-carrying groups. This allows for the analysis of the distance dependence of the D-parameter as well as the through-bond and through-space spin-spin interaction. From these results we established the limitations of the point-dipole approximation. The results of this analysis demonstrate that even very modest amounts of spin delocalization can cause significant deviations from pure point-dipole behavior and consequently cause the EPR derived distances to deviate from the N-O midpoint distance by up to several Ångström. If unsaturated linkers are used, the distance dependence of D does not follow the inverse cubic behavior predicted by the point dipole model. However, for commonly-used non-aromatic nitroxide rings connected by a saturated linker, the point dipole approximation works well. Among the various point dipole variants tested in this work for delocalized spins, the most successful one is based on distributed point dipoles with spin populations derived from quantum chemical calculations. The distance dependence of the isotropic Heisenberg exchange parameter J has also been studied theoretically. The decay was found to be monoexponential with a decay constant of ~1 Å−1. Thus at linker lengths between 6–8 carbon atoms between a nitroxide radical pair a switch from the strong to the weak exchange limit is predicted.

Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.Cellular processes including signal transduction, vesicular trafficking, and cytoskeletal rearrangement require selective recruitment of proteins to membrane surfaces. Well established mechanisms for localizing cytosolic proteins to membranes include electrostatic interactions through a basic peptide sequence, anchoring by covalently attached acyl chains, and association with the cytoplasmic domains of transmembrane proteins (reviewed in Refs. 1 and 2). (4), PDZ (5), and PTB (6) domains. Although the majority of these domains can interact with several PIs, the FYVE domain is remarkably selective for PtdIns(3)P (7-9). In addition to PI ligation, these domains often insert hydrophobic elements into the membrane bilayer, as has been demonstrated for the C2 (10), ENTH (11), FERM (12), FYVE (13), and PX (14) domains and the vinculin tail (15). Insertion into the membrane can be accompanied by interactions with multiple lipid head groups. For example, the PX domain of the p47 subunit of the NADPH oxidase binds cooperatively to PtdIns(3)P and phosphatidic acid (16), and the vinculin tail co-ligates phosphatidylinositol 4,5-bisphosphate and PtdSer (15). Although it is becoming evident that insertion of proteins into membranes is widespread, the three-dimensional orientations and quantitative binding properties remain challenging to characterize. The most common electron paramagnetic resonance and fluorescence approaches have provided important insights (17-20) but require covalent attachment of paramagnetic groups to various positions of the protein or mutations of residues. The inevitable effects of these modifications on lipi...

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