B. Scott Perrin

High-resolution structures and orientations of antimicrobial peptides piscidin 1 and piscidin 3 in fluid bilayers reveal tilting, kinking, and bilayer immersion.

While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of 1H–15N dipolar couplings and 15N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (∼25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ∼1.2–3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.

Web-Based Computational Chemistry Education with CHARMMing III: Reduction Potentials of Electron Transfer Proteins

A module for fast determination of reduction potentials, E°, of redox-active proteins has been implemented in the CHARMM INterface and Graphics (CHARMMing) web portal (www.charmming.org). The free energy of reduction, which is proportional to E°, is composed of an intrinsic contribution due to the redox site and an environmental contribution due to the protein and solvent. Here, the intrinsic contribution is selected from a library of pre-calculated density functional theory values for each type of redox site and redox couple, while the environmental contribution is calculated from a crystal structure of the protein using Poisson-Boltzmann continuum electrostatics. An accompanying lesson demonstrates a calculation of E°. In this lesson, an ionizable residue in a [4Fe-4S]-protein that causes a pH-dependent E° is identified, and the E° of a mutant that would test the identification is predicted. This demonstration is valuable to both computational chemistry students and researchers interested in predicting sequence determinants of E° for mutagenesis.

Structural properties of apolipoprotein A-I mimetic peptides that promote ABCA1-dependent cholesterol efflux

Peptides mimicking the major protein of highdensity lipoprotein (HDL), apolipoprotein A-I (apoA-I), are promising therapeutics for cardiovascular diseases. Similar to apoA-I, their atheroprotective property is attributed to their ability to form discoidal HDL-like particles by extracting cellular cholesterol and phospholipids from lipid microdomains created by the ABCA1 transporter in a process called cholesterol efflux. The structural features of peptides that enable cholesterol efflux are not well understood. Herein, four synthetic amphipathic peptides denoted ELK, which only contain Glu, Leu, Lys, and sometimes Ala, and which have a wide range of net charges and hydrophobicities, were examined for cholesterol efflux. Experiments show that ELKs with a net neutral charge and a hydrophobic face that subtends an angle of at least 140° are optimal for cholesterol efflux. All-atom molecular dynamics simulations show that peptides that are effective in promoting cholesterol efflux stabilize HDL nanodiscs formed by these peptides by the orderly covering of the hydrophobic acyl chains on the edge of the disc. In contrast to apoA-I, which forms an anti-parallel double belt around the HDL, active peptides assemble in a mostly anti-parallel “picket fence” arrangement. These results shed light on the efflux ability of apoA-I mimetics and inform the future design of such therapeutics.

Identifying Residues That Cause pH-Dependent Reduction Potentials

The pH dependence of the reduction potential E° for a metalloprotein indicates that the protonation state of at least one residue near the redox site changes and may be important for its activity. The responsible residue is usually identified by site-specific mutagenesis, which may be time-consuming. Here, the titration of E° for Chromatium vinosum high-potential iron-sulfur protein is predicted to be in good agreement with experiment using density functional theory and Poisson-Boltzmann calculations if only the sole histidine undergoes changes in protonation. The implementation of this approach into CHARMMing, a user-friendly web-based portal, allows users to identify residues in other proteins causing similar pH dependence.

Identification of a novel lipid binding motif in apolipoprotein B by the analysis of hydrophobic cluster domains

Apolipoprotein B (apoB) is a large amphipathic protein that is the structural scaffold for the formation of several classes of lipoproteins involved in lipid transport throughout the body. The goal of the present study was to identify specific domains in the apoB sequence that contribute to its lipid binding properties. A sequence analysis algorithm was developed to identify stretches of hydrophobic amino acids devoid of charged amino acids, which are referred to as hydrophobic cluster domains (HCDs). This analysis identified 78 HCDs in apoB with hydrophobic stretches ranging from 6 to 26 residues. Each HCD was analyzed in silico for secondary structure and lipid binding properties, and a subset was synthesized for experimental evaluation. One HCD peptide, B38, showed high affinity binding to both isolated HDL and LDL, and could exchange between lipoproteins. All-atom molecular dynamics simulations indicate that B38 inserts 3.7Å below the phosphate plane of the bilayer. B38 forms an unusual α-helix with a broad hydrophobic face and polar serine and threonine residues on the opposite face. Based on this structure, we hypothesized that B38 could efflux cholesterol from cells. B38 showed a 12-fold greater activity than the 5A peptide, a bihelical Class A amphipathic helix (EC50 of 0.2658 vs. 3.188μM; p<0.0001), in promoting cholesterol efflux from ABCA1 expressing BHK-1 cells. In conclusion, we have identified novel domains within apoB that contribute to its lipid biding properties. Additionally, we have discovered a unique amphipathic helix design for efficient ABCA1-specific cholesterol efflux.

CHAPTER 2:Combining NMR Spectroscopic Measurements and Molecular Dynamics Simulations to Determine the Orientation of Amphipathic Peptides in Lipid Bilayers

Determining the orientation of an amphipathic α-helix in a lipid bilayer can be achieved indirectly with oriented sample solid-state NMR or directly by molecular dynamics (MD) simulations. In a peptide plane, both the 1H–15N dipolar coupling (DC) and 15N chemical shift (CS) tensors are sensitive to orientation with respect to the static magnetic field. Two-dimensional separated local field spectra from α-helices display ellipsoidal patterns called PISA wheels. Plotting DNH or CS values from these spectra as a function of residue number produces sinusoidal waves. PISA wheels, dipolar waves and CS waves each give the orientation of the α-helix in the bilayer; however, this is not always accurate. A bias in orientation due to dynamic fluctuations in tilt was demonstrated for transmembrane peptides studied using PISA wheels. Here, the ambiguity associated with the use of dipolar and CS waves is presented for piscidin, an antimicrobial peptide. The dipolar and CS wave-fit analysis resulted in multiple solutions for the peptide orientation and MD was used to identify the more probable value. Since the NMR observables can be affected by peptide dynamics, the effect on the NH bond order parameter is explored and the implications for determination of peptide structures are discussed.

Characterization of Apolipoprotein Mimetic Peptides on Nascent High Density Lipoproteins

A nascent high-density lipoprotein (HDL) is a discoidal bilayer composed of phospholipids, cholesterols, and two apolipoproteins that form a scaffold that stabilizes the assembly. Apolipoprotein mimetic peptides are short synthetic peptides that share features of apolipoproteins and have potential therapeutic value based on their ability to form and stabilize nascent HDL. A key question in designing mimetic peptides is why some are more efficient than others. We characterize the properties of four mimetic peptides rich in the amino acids E, L, and K (called ELK peptides), using a combination of Molecular Dynamics simulations and experimental techniques. Experiments show that the hydrophobic and neutral ELKs have a significantly higher ability to form nascent HDLs than the positive or negative peptides. An in silico model of a discoidal bilayer was developed by introducing a water slab perpendicular to the bilayer head group surface, leading to acyl chains of edges exposed to water. Simulations on this model discoidal bilayer with peptides indicate that hydrophobic and neutral ELKs stabilize the assembly by forming scaffolds at the edges in a picket fence arrangement; the adjacent peptides are held together by strong salt-bridges. In contrast, the positive and negative ELKs diffuse to the head group surface, do not form effective scaffolds, and fail to stabilize the discoidal assembly. Hence, the simulations provide a structural rational for the experimental observations, and provide an avenue for computer based design of apolipoprotein mimetic peptides.

Protein dynamics and the all-ferrous [Fe4 S4 ] cluster in the nitrogenase iron protein.

In nitrogen fixation by Azotobacter vinelandii nitrogenase, the iron protein (FeP) binds to and subsequently transfers electrons to the molybdenum–FeP, which contains the nitrogen fixation site, along with hydrolysis of two ATPs. However, the nature of the reduced state cluster is not completely clear. While reduced FeP is generally thought to contain an [Fe4S4]1+ cluster, evidence also exists for an all‐ferrous [Fe4S4]0 cluster. Since the former indicates a single electron is transferred per two ATPs hydrolyzed while the latter indicates two electrons could be transferred per two ATPs hydrolyzed, an all‐ferrous [Fe4S4]0 cluster in FeP is potenially two times more efficient. However, the 1+/0 reduction potential has been measured in the protein at both 460 and 790 mV, causing the biological significance to be questioned. Here, “density functional theory plus Poisson Boltzmann” calculations show that cluster movement relative to the protein surface observed in the crystal structures could account for both measured values. In addition, elastic network mode analysis indicates that such movement occurs in low frequency vibrations of the protein, implying protein dynamics might lead to variations in reduction potential. Furthermore, the different reductants used in the conflicting measurements of the reduction potential could be differentially affecting the protein dynamics. Moreover, even if the all‐ferrous cluster is not the biologically relevant cluster, mutagenesis to stabilize the conformation with the more exposed cluster may be useful for bioengineering more efficient enzymes.