Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small angle x-ray scattering to investigate interactions between heterogeneous membranes and members of the defensin subfamilies, α-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay (‘negative Gaussian’) membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, since it is a necessary condition for processes such as pore formation, blebbing, budding, vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This ‘design rule’ is consistent with the amino acid compositions of 1,080 known cationic AMPs.

We have used ab initio quantum chemical techniques to compute the (13)C(alpha) and (13)C(beta) shielding surfaces for the 14 amino acids not previously investigated (R. H. Havlin et al., J. Am. Chem. Soc. 1997, 119, 11951-11958) in their most popular conformations. The spans (Omega = sigma(33) - sigma(11)) of all the tensors reported here are large ( approximately 34 ppm) and there are only very minor differences between helical and sheet residues. This is in contrast to the previous report in which Val, Ile and Thr were reported to have large ( approximately 12 ppm) differences in Omega between helical and sheet geometries. Apparently, only the beta-branched (beta-disubstituted) amino acids have such large CSA span (Omega) differences; however, there are uniformly large differences in the solution-NMR-determined CSA (Deltasigma = sigma(orth) - sigma(par)) between helices and sheets in all amino acids considered. This effect is overwhelmingly due to a change in shielding tensor orientation. With the aid of such shielding tensor orientation information, we computed Deltasigma values for all of the amino acids in calmodulin/M13 and ubiquitin. For ubiquitin, we find only a 2.7 ppm rmsd between theory and experiment for Deltasigma over an approximately 45 ppm range, a 0.96 slope, and an R(2) = 0.94 value when using an average solution NMR structure. We also report C(beta) shielding tensor results for these same amino acids, which reflect the small isotropic chemical shift differences seen experimentally, together with similar C(beta) shielding tensor magnitudes and orientations. In addition, we describe the results of calculations of C(alpha), C(beta), C(gamma)1, C(gamma)2, and C(delta) shifts in the two isoleucine residues in bovine pancreatic trypsin inhibitor and the four isoleucines in a cytochrome c and demonstrate that the side chain chemical shifts are strongly influenced by chi(2) torsion angle effects. There is very good agreement between theory and experiment using either X-ray or average solution NMR structures. Overall, these results show that both C(alpha) backbone chemical shift anisotropy results as well as backbone and side chain (13)C isotropic shifts can now be predicted with good accuracy by using quantum chemical methods, which should facilitate solution structure determination/refinement using such shielding tensor surface information.

We have investigated the carbon-13 solution nuclear magnetic resonance (NMR) chemical shifts of Cα, Cβ, and Cγ carbons of 19 valine residues in a vertebrate calmodulin, a nuclease from Staphylococcus aureus, and a ubiquitin. Using empirical chemical shift surfaces to predict Cα, Cβ shifts from known, X-ray φ,ψ values, we find moderate accord between prediction and experiment. Ab initio calculations with coupled Hartree−Fock (HF) methods and X-ray structures yield poor agreement with experiment. There is an improvement in the ab initio results when the side chain χ1 torsion angles are adjusted to their lowest energy conformers, using either ab initio quantum chemical or empirical methods, and a further small improvement when the effects of peptide-backbone charge fields are introduced. However, although the theoretical and experimental results are highly correlated (R 2 ∼ 0.90), the observed slopes of ∼−0.6−0.8 are less than the ideal value of −1, even when large uniform basis sets are used. Use of density functional theory (DFT) methods improves the quality of the predictions for both Cα (slope = −1.1, R 2 = 0.91) and Cβ (slope = −0.93, R 2 = 0.89), as well as giving moderately good results for Cγ. This effect is thought to arise from a small, conformationally-sensitive contribution to shielding arising from electron correlation. Additional shielding calculations on model compounds reveal similar effects. Results for valine residues in interleukin-1β are less highly correlated, possibly due to larger crystal−solution structural differences. When taken together, these results for 19 valine residues in 3 proteins indicate that choosing the lowest energy χ1 conformer together with X-ray φ,ψ values enables the successful prediction of both Cα and Cβ shifts, with DFT giving close to ideal slopes and R 2 values between theory and experiment. These results strongly suggest that the most highly populated valine side-chain conformers are those having the lowest (computationally determined) energy, as evidenced by the ability to predict essentially all Cα, Cβ chemical shifts in calmodulin, SNase, and ubiquitin, as well as moderate accord for Cγ. These observations suggest a role for chemical shifts and energy minimization/geometry optimization in the refinement of protein structures in solution, and potentially in the solid state as well.

We have synthesized and studied via solid-state NMR, Mössbauer spectroscopy, single-crystal X-ray diffraction, and density functional theory the following Fe−O2 analogue metalloporphyrins:  Fe(5,10,15,20-tetraphenylporphyrinate) (nitrosobenzene)(1-methylimidazole); Fe(5,10,15,20-tetraphenylporphyrinate) (nitrosobenzene)(pyridine); Fe(5,10,15,20-tetraphenylporphyrinate)(4-nitroso-N,N-dimethylaniline)(pyridine); Fe(2,3,7,8,12,13,17,18-octaethylporphyrinate) (nitrosobenzene)(1-methylimidazole) and Co(2,3,7,8,12,13,17,18-octaethylporphyrinate)(NO). Our results show that the porphyrin rings of the two tetraphenylporphyrins containing pyridine are ruffled while the other three compounds are planar:  reasons for this are discussed. The solid-state NMR and Mössbauer spectroscopic results are well reproduced by the DFT calculations, which then enable the testing of various models of Fe−O2 bonding in metalloporphyrins and metalloproteins. We find no evidence for two binding sites in oxypicket fence porphyrin, characterized by very different electric field gradients. However, the experimental Mössbauer quadrupole splittings can be readily accounted for by fast axial rotation of the Fe−O2 unit. Unlike oxymyoglobin, the Mössbauer quadrupole splitting in PhNO•myoglobin does not change with temperature, due to the static nature of the Fe•PhNO subunit, as verified by 2H NMR of Mb•[2H5]PhNO. Rotation of O2 to a second (minority) site in oxymyoglobin can reduce the experimental quadrupole splittings, either by simple exchange averaging, or by an electronic mechanism, without significant changes in the Fe−O−O bond geometry, or a change in sign of the quadrupole splitting. DFT calculations of the molecular electrostatic potentials in CO, PhNO, and O2-metalloporphyrin complexes show that the oxygen sites in the PhNO and O2 complexes are more electronegative than that in the CO system, which strongly supports the idea that hydrogen bonding to O2 will be a major contributor to O2/CO discrimination in heme proteins.

We have carried out a solid-state magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic investigation of the (13)C(alpha) chemical shielding tensors of alanine, valine, and leucine residues in a series of crystalline peptides of known structure. For alanine and leucine, which are not branched at the beta-carbon, the experimental chemical shift anisotropy (CSA) spans (Omega) are large, about 30 ppm, independent of whether the residues adopt helical or sheet geometries, and are in generally good accord with Omega values calculated by using ab initio Hartree-Fock quantum chemical methods. The experimental Omegas for valine C(alpha) in two peptides (in sheet geometries) are also large and in good agreement with theoretical predictions. In contrast, the "CSAs" (Deltasigma) obtained from solution NMR data for alanine, valine, and leucine residues in proteins show major differences, with helical residues having Deltasigma values of approximately 6 ppm while sheet residues have Deltasigma approximately 27 ppm. The origins of these differences are shown to be due to the different definitions of the CSA. When defined in terms of the solution NMR CSA, the solid-state results also show small helical but large sheet CSA values. These results are of interest since they lead to the idea that only the beta-branched amino acids threonine, valine, and isoleucine can have small (static) tensor spans, Omega (in helical geometries), and that the small helical "CSAs" seen in solution NMR are overwhelmingly dominated by changes in tensor orientation, from sheet to helix. These results have important implications for solid-state NMR structural studies which utilize the CSA span, Omega, to differentiate between helical and sheet residues. Specifically, there will be only a small degree of spectral editing possible in solid proteins since the spans, Omega, for the dominant nonbranched amino acids are quite similar. Editing on the basis of Omega will, however, be very effective for many Thr, Val, and Ileu residues, which frequently have small ( approximately 15-20 ppm) helical CSA (Omega) spans.