Phospholipid lateral diffusion in the presence of cationic peptides as measured via 31P CODEX NMR.

The effects of two cationic peptides on phospholipid lateral diffusion in binary mixtures of POPC with various anionic phospholipids were measured via 31P CODEX NMR. Large unilamellar vesicles composed of POPC/POPG (70/30 mol/mol), or POPC/DOPS (70/30 mol/mol), or POPC/TOCL (85/15 mol/mol), or POPC/DOPA (50/50 mol/mol) were exposed to either polylysine (pLYS, N = 134 monomers) or KL-14 (KKLL KKAKK LLKKL), a model amphipathic helical peptide, in an amount corresponding to 80% neutralization of the anionic phospholipid charge by the cationic lysine residues. In the absence of added peptide, phospholipid lateral diffusion coefficients (all measured at 10 °C) increased with increasing reduced temperature (T-Tm). The POPC/DOPA mixture was an exception to this generalization, in that lateral diffusion for both components was far slower than any other mixture investigated, an effect attributed to intermolecular hydrogen bonding. The addition of pLYS or KL-14 decreased lateral diffusion in the POPC/DOPS LUV, but had minimal effects in the POPC/POPG LUV, indicating that ease of access of the cationic peptide residues to the anionic phospholipid groups was important. Both cationic peptides produced the opposite effect in the POPC/DOPA case, in that lateral diffusion increased significantly in their presence, with KL-14 being most effective. This latter observation was interpreted in terms of the electrostatic / H-bond model proposed by Kooijman et al. [Journal of Biological Chemistry, 282:11356-11,364, 2007] to describe the mechanism of interaction between the phosphomonoester head group of PA and the tertiary amine of lysine.

ProxyPhos sensors for the detection of negatively charged membranes.

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.

Direct quantitative 13 C-filtered 1 H magnetic resonance imaging of PEGylated biomacromolecules in vivo.

PURPOSE: 1 H MRI is an established diagnostic method that generally relies on detection of water. Imaging specific macromolecules is normally accomplished only indirectly through the use of paramagnetic tags, which alter the water signal in their vicinity. We demonstrate a new approach in which macromolecular constituents, such as proteins and drug delivery systems, are observed directly and quantitatively in vivo using 1 H MRI of 13 C-labeled poly(ethylene glycol) (13 C-PEG) tags. METHODS: Molecular imaging of 13 C-PEG-labeled species was accomplished by incorporating a modified heteronuclear multiple quantum coherence filter into a gradient echo imaging sequence. We demonstrate the approach by monitoring the real-time distribution of 13 C-PEG and 13 C-PEGylated albumin injected into the hind leg of a mouse. RESULTS: Filtering the 1 H PEG signal through the directly coupled 13 C nuclei largely eliminates background water and fat signals, thus enabling the imaging of molecules using 1 H MRI. CONCLUSION: PEGylation is widely employed to enhance the performance of a multitude of macromolecular therapeutics and drug delivery systems, and 13 C-filtered 1 H MRI of 13 C-PEG thus offers the possibility of imaging and quantitating their distribution in living systems in real time. Magn Reson Med 77:1553-1561, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Quantitative Detection of PEGylated Biomacromolecules in Biological Fluids by NMR.

The accumulation, biodistribution, and clearance profiles of therapeutic agents are key factors relevant to their efficacy. Determining these properties constitutes an ongoing experimental challenge. Many such therapeutics, including small molecules, peptides, proteins, tissue scaffolds, and drug delivery vehicles, are conjugated to poly(ethylene glycol) (PEG) as this improves their bioavailability and in vivo stability. We demonstrate here that (1)H NMR spectroscopy can be used to quantify PEGylated species in complex biological fluids directly, rapidly, and with minimal sample preparation. PEG bears a large number of spectroscopically equivalent protons exhibiting a narrow NMR line width while resonating at a (1)H NMR frequency distinct from most other biochemical signals. We demonstrate that PEG provides a robust signal allowing detection of concentrations as low as 10 µg/mL in blood. This PEG detection limit is lowered by another order of magnitude when background proton signals are minimized using (13)C-enriched PEG in combination with a double quantum filter to remove (1)H signals from non-(13)C-labeled species. Quantitative detection of PEG via these methods is shown in pig blood and goat serum as examples of complex biological fluids. More practically, we quantify the blood clearance of (13)C-PEG and PEGylated-BSA (bovine serum albumin) following their intravenous injection in live rats. Given the relative insensitivity of line width to PEG size, we anticipate that the biodistribution and clearance profiles of virtually any PEGylated biomacromolecule from biological fluid samples can be routinely measured by (1)H NMR without any filtering or treatment steps.

Shoulder Arthroscopy Does Not Adequately Visualize Pathology of the Long Head of Biceps Tendon.

BACKGROUND: Pulling the long head of the biceps tendon into the joint at arthroscopy is a common method for evaluation of tendinopathic lesions. However, the rate of missed diagnoses when using this technique is reported to be as high as 30% to 50%. HYPOTHESIS: Tendon excursion achieved using a standard arthroscopic probe does not allow adequate visualization of extra-articular sites of predilection of tendinopathy. STUDY DESIGN: Descriptive laboratory study. METHODS: Seven forequarter amputation cadaveric specimens were evaluated. The biceps tendon was tagged to mark the intra-articular length and the maximum excursions achieved using a probe and a grasper in both beach-chair and lateral positions. Statistical analyses were performed using analysis of variance to compare means. RESULTS: The mean intra-articular and extra-articular lengths of the tendons were 23.9 and 82.3 mm, respectively. The length of tendon that could be visualized by pulling it into the joint with a probe through the anterior midglenoid portal was not significantly different when using either lateral decubitus (mean ± SD, 29.9 ± 3.89 mm; 95% CI, 25.7-34 mm) or beach-chair positions (32.7 ± 4.23 mm; 95% CI, 28.6-36.8 mm). The maximum length of the overall tendon visualized in any specimen using a standard technique was 37 mm. Although there was a trend to greater excursion using a grasper through the same portal, this was not statistically significant. However, using a grasper through the anterosuperior portal gave a significantly greater mean excursion than any other technique (46.7 ± 4.31 mm; 95% CI, 42.6-50.8 mm), but this still failed to allow evaluation of Denard zone C. CONCLUSION: Pulling the tendon into the joint with a probe via an anterior portal does not allow visualization of distal sites of predilection of pathology. Surgeons should be aware that this technique is inadequate and can result in missed diagnoses. CLINICAL RELEVANCE: This study demonstrates that glenohumeral arthroscopy does not allow visualization of common areas of pathology of the long head of the biceps tendon.

A Molecular Rotor Possessing an H-M-H "Spoke" on a P-M-P "Axle": A Platinum(II) trans-Dihydride Spins Rapidly Even at 75 K.

A new class of low-barrier molecular rotors, metal trans-dihydrides, is suggested here. To test whether rapid rotation can be achieved, the known complex trans-H2Pt(P(t)Bu3)2 was experimentally studied by (2)H and (195)Pt solid-state NMR spectroscopy (powder pattern changes with temperature) and computationally modeled as a (t)Bu3P-Pt-P(t)Bu3 stator with a spinning H-Pt-H rotator. Whereas the related chloro-hydride complex, trans-H(Cl)Pt(P(t)Bu3)2, does not show rotational behavior at room temperature, the dihydride trans-H2Pt(P(t)Bu3)2 rotates fast on the NMR time scale, even at low temperatures down to at least 75 K. The highest barrier to rotation is estimated to be ∼3 kcal mol(-1), for the roughly 3 Šlong rotator in trans-H2Pt(P(t)Bu3)2.

Centerband-only-detection-of-exchange (31)P nuclear magnetic resonance and phospholipid lateral diffusion: theory, simulation and experiment.

Centerband-only-detection-of-exchange (CODEX) (31)P NMR lateral diffusion measurements were performed on dimyristoylphosphatidylcholine (DMPC) assembled into large unilamellar spherical vesicles. Optimization of sample and NMR acquisition conditions provided significant sensitivity enhancements relative to an earlier first report (Q. Saleem, A. Lai, H. Morales, and P. M. Macdonald, Chem. Phys. Lipids, 2012, 165, 721). An analytical description was developed that permitted the extraction of lateral diffusion coefficients from CODEX data, based on a Gaussian-diffusion-on-a-sphere model (A. Ghosh, J. Samuel, and S. Sinha, Europhys. Lett., 2012, 98, 30003-p1) as relevant to CODEX (31)P NMR measurements on a population of spherical unilamellar phospholipid bilayer vesicles displaying a distribution of vesicle radii.

Single lipid bilayer deposition on polymer surfaces using bicelles.

A lipid bilayer was deposited on a 3 µm diameter polystyrene (PS) bead via hydrophobic anchoring of bicelles containing oxyamine-bearing cholesteric moieties reacting with the aldehyde functionalized bead surface. Discoidal bicelles were formed by mixing dimyristoylphosphatidylcholine (DMPC), dihexanoylphosphatidylcholine (DHPC), dimyristoyltrimethylammonium propane (DMTAP), and the oxyamine-terminated cholesterol derivative, cholest-5-en-3ß-oxy-oct-3,6-oxa-an-8-oxyamine (CHOLOA), in the molar ratio DMPC/DHCP/DMTAP/CHOLOA (1/0.5/0.01/0.05) in water. Upon exposure to aldehyde-bearing PS beads, a stable single lipid bilayer coating rapidly formed at the bead surface. Fluorescence recovery after photobleaching demonstrated that the deposited lipids fused into an encapsulating lipid bilayer. Electrospray ionization mass spectrometry showed that the short chain lipid DHPC was entirely absent from the PS adherent lipid coating. Fluorescence quenching measurements proved that the coating was a single lipid bilayer. The bicelle coating method is thus simple and robust, can be modified to include membrane-associated species, and can be adapted to coat any number of different surfaces.

Thermal stabilization of bicelles by a bile-salt-derived detergent: a combined ³¹P and ²H nuclear magnetic resonance study.

The properties of bicelles composed of mixtures of long-chain lipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG), stabilized by zwitterionic bile salt analogue 3-[(3-cholamidopropyl)dimethyl-d6-ammonio]-2-hydroxy-1-propanesulfonate (CHAPSO-d6), deuterated at both amino methyls, were investigated by a combination of (31)P and (2)H NMR, focusing on the behavior of CHAPSO as a function of temperature. For compositions of molar ratio q = [DMPC + DMPG]/[CHAPSO] = 3, R = [DMPG]/[DMPC + DMPG] = 0, 0.01 and 0.10 and lipid concentration CL = 25 wt % lipid at temperatures of between 30 and 60 °C, magnetic alignment was readily achieved as assessed via both (31)P NMR of the phospholipids and (2)H NMR of CHAPSO-d6. Increasing temperature yielded higher values for the chemical shift anisotropy of the former and the quadrupole splitting of the latter, consistent with the progressive migration of CHAPSO from edge regions into planar regions of the bicellar assemblies. However, relative to dihexadecyl phosphatidylcholine (DHPC), CHAPSO exhibited lower miscibility with DMPC, although the presence of DMPG enhanced this miscibility. At 65 °C, thermal instability became evident in the appearance of a separate isotropic component in both (31)P and (2)H NMR spectra. This isotropic phase was CHAPSO-enriched but less so as a function of increasing DMPG. These findings indicate that the enhanced thermal stability of CHAPSO- versus DHPC-containing bicelles arises from a combination of the larger surface area that edge CHAPSO is able to mask, mole for mole, and its relative preference for edge regions, plus, possibly, specific interactions with DMPG.

Temperature and pressure based NMR studies of detergent micelle phase equilibria.

Bulk thermodynamic and volumetric parameters (ΔGmic°, ΔHmic°, ΔSmic°, ΔCp,mic°, ΔVmic°, and Δκmic°) associated with the monomer­micelle equilibrium, were directly determined for a variety of common detergents [sodium n-dodecyl sulfate (SDS), n-dodecyl phosphocholine (DPC), n-dodecyl-ß-d-maltoside (DDM), and 7-cyclohexyl-1-heptyl phosphocholine (CyF)] via 1H NMR spectroscopy. For each temperature and pressure point, the critical micelle concentration (cmc) was obtained from a single 1H NMR spectrum at a single intermediate concentration by referencing the observed chemical shift to those of pure monomer and pure micellar phases. This permitted rapid measurements of the cmc over a range of temperatures and pressures. In all cases, micelle formation was strongly entropically favored, while enthalpy changes were all positive, with the exception of SDS, which exhibited a modestly negative enthalpy of micellization. Heat capacity changes were also characteristically negative, while partial molar volume changes were uniformly positive, as expected for an aggregation process dictated by hydrophobic effects. Isothermal compressibility changes were found to be consistent with previous measurements using other techniques. Thermodynamic measurements were also related to spectroscopic studies of topology and micelle structure. For example, paramagnetic effects resulting from the addition of dioxygen provided microscopic topological details concerning the hydrophobicity gradient along the detergent chains within their respective micelles as detected by 1H NMR. In a second example, combined 13C and 1H NMR chemical shift changes arising from application of high pressure, or upon micellization, of CyF provided site-specific details regarding micelle topology. In this fashion, bulk thermodynamics could be related to microscopic topological details within the detergent micelle.

Liposome-coated hydrogel spheres: delivery vehicles with tandem release from distinct compartments.

We have fabricated unilamellar lipid bilayer VESicle-COated hydrogel spheres (VESCOgels) by carbodiimide-mediated coupling of liposomes bearing surface amines to core-shell hydrogel spheres bearing surface carboxyls. The amine-containing moiety, 3-O (2-aminoethoxyethyloxyethyl)carbamyl cholesterol (AECHO), was incorporated into large unilamellar vesicles (LUVs), diameter ∼100 nm, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The hydrogel, diameter ∼ 1 µm, consisted of a core of poly(N-isopropyl acrylamide) (pNIPAM) and a shell of p(NIPAM-co-acrylic acid (AA)). Activation of these surface-displayed carboxyls with N-hydroxysuccinimidyl (NHS) esters permitted amine coupling upon addition of AECHO-containing POPC LUVs. Bilayer integrity of the hydrogel-bound LUVs was maintained, and fusion of LUVs did not occur. Fluorescence assays of the release of cobalt-calcein trapped within hydrogel-bound LUVs and of sodium fluorescein trapped within the hydrogel itself showed that each compartment retained its distinct release attributes: fast release from the microgel and slow release from the LUVs. It is envisioned that VESCOgels will be useful, therefore, in applications requiring temporally controlled delivery of distinct drugs.

Morphological characterization of DMPC/CHAPSO bicellar mixtures: a combined SANS and NMR study.

Spontaneously forming structures of a system composed of dimyristoyl phosphatidylcholine (DMPC) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) were studied by small-angle neutron scattering (SANS), (31)P NMR, and stimulated echo (STE) pulsed field gradient (PFG) (1)H NMR diffusion measurements. Charged lipid dimyristoyl phosphatidylglycerol (DMPG) was used to induce different surface charge densities. The structures adopted were investigated as a function of temperature and lipid concentration for samples with a constant molar ratio of long-chain to short-chain lipids (= 3). In the absence of DMPG, zwitterionic bicellar mixtures exhibited a phase transition from discoidal bicelles, or ribbons, to multilamellar vesicles either upon dilution or with increased temperature. CHAPSO-containing mixtures showed a higher thermal stability in morphology than DHPC-containing mixtures at the corresponding lipid concentrations. In the presence of DMPG, discoidal bicelles (or ribbons) were also found at low temperature and lower lipid concentration mixtures. At high temperature, perforated lamellae were observed in high-concentration mixtures (≥7.5 wt %) whereas uniform unilamellar vesicles and bicelles formed in low-concentration mixtures (≤2.5 wt %), respectively, when the mixtures were moderately and highly charged. From the results, spontaneous structural diagrams of the zwitterionic and charged systems were constructed.

NMR methods for measuring lateral diffusion in membranes.

Lateral diffusion is a fundamental property of biological membrane components, important for a host of biomembrane functions. Although long studied, novel aspects of the relationship between the structure of membrane components and their lateral diffusion properties continue to emerge. NMR-based lateral diffusion measurements are complicated by the spectral broadening arising from the slow anisotropic motions in membranes. Nevertheless, both pulsed field gradient (PFG) and exchange spectroscopy (EXSY) methods can be adapted to permit NMR measurements of lateral diffusion in membranes. These variously will be described in overview, highlighting advantages and limitations of each, but with particular emphasis on results from our laboratory using (1)H PFG NMR measurements in magnetically aligned bicelles and (31)P CODEX (Centreband-Only-Detection-of-Exchange) measurements in spherical phospholipid vesicles.

Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway.

Membrane proteins constitute a significant fraction of the proteome and are important drug targets. While the transmembrane (TM) segments of these proteins are primarily composed of hydrophobic residues, the inclusion of polar residues-either naturally occurring or as a consequence of a disease-related mutation-places a significant folding burden in this environment, potentially impacting bilayer insertion and/or association of neighboring TM helices. Here we investigate the role of an anionic detergent, sodium dodecylsulfate (SDS), and a zwitterionic detergent, dodecylphosphocholine (DPC), in the folding process, and the effects induced by a single polar substitution, on structure and topology of model α-helical TM segments. The peptides, represented by KK-YAAAIAAIAWAXAAIAAAIAA-KKK-NH(2), where X is I or N, are designed with high aqueous solubilities, through poly-lysine tags. Circular dichroism (CD) and NMR were used to monitor peptide secondary structure and diffusional mobility of both peptide and the detergent hosts. For both peptides, SDS binding commenced at a concentration below its CMC, due to Coulombic attraction of anionic SDS to cationic Lys residues. Increasing SDS binding correlated with increasing peptide helicity. Pulsed field gradient (PFG) NMR diffusion measurements revealed that the Asn-containing peptide bound four fewer detergent molecules, corresponding to ca. 20% less SDS than bound by the Ile peptide. Conversely, zwitterionic DPC binding to either peptide was not observed until the DPC concentration approached its CMC. Our findings confirm quantitatively that a single polar residue within a TM segment may have a significant influence on its local membrane environment.

Lateral diffusion of bilayer lipids measured via (31)P CODEX NMR.

We have employed (31)P CODEX (centre-band-only-detection-of-exchange) NMR to measure lateral diffusion coefficients of phospholipids in unilamellar lipid bilayer vesicles consisting of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), alone or in mixtures with 30 mol% 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) or cholesterol (CHOL). The lateral diffusion coefficients of POPC and POPG were extracted from experimental CODEX signal decays as a function of increasing mixing time, after accounting for the vesicle's size and size distribution, as determined via dynamic light scattering, and the viscosity of the vesicular suspension, as determined via (1)H pulsed field gradient NMR. Lateral diffusion coefficients for POPC and POPG determined in this fashion fell in the range 1.0-3.2 × 10(-12) m(2) s(-1) at 10 °C, depending on the vesicular composition, in good agreement with accepted values. Thus, two advantages of (31)P CODEX NMR for phospholipid lateral diffusion measurements are demonstrated: no labelling of the molecule of interest is necessary, and multiple lateral diffusion coefficients can be measured simultaneously. It is expected that this approach will prove particularly useful in diagnosing heterogeneities in lateral diffusion behaviours, such as might be expected for specific lipid-lipid or lipid-protein interactions, and thermotropic or electrostatically induced phase inhomogeneities.

Site specific interaction of the polyphenol EGCG with the SEVI amyloid precursor peptide PAP(248-286).

Recently, a 39 amino acid peptide fragment from prostatic acid phosphatase has been isolated from seminal fluid that can enhance infectivity of the HIV virus by up to 4-5 orders of magnitude. PAP(248-286) is effective in enhancing HIV infectivity only when it is aggregated into amyloid fibers termed SEVI. The polyphenol EGCG (epigallocatechin-3-gallate) has been shown to disrupt both SEVI formation and HIV promotion by SEVI, but the mechanism by which it accomplishes this task is unknown. Here, we show that EGCG interacts specifically with the side chains of monomeric PAP(248-286) in two regions (K251-R257 and N269-I277) of primarily charged residues, particularly lysine. The specificity of interaction to these two sites is contrary to previous studies on the interaction of EGCG with other amyloidogenic proteins, which showed the nonspecific interaction of EGCG with exposed backbone sites of unfolded amyloidogenic proteins. This interaction is specific to EGCG as the related gallocatechin (GC) molecule, which shows greatly decreased antiamyloid activity, exhibits minimal interaction with monomeric PAP(248-286). The EGCG binding was shown to occur in two steps, with the initial formation of a weakly bound complex followed by a pH dependent formation of a tightly bound complex. Experiments in which the lysine residues of PAP(248-286) have been chemically modified suggest the tightly bound complex is created by Schiff-base formation with lysine residues. The results of this study could aid in the development of small molecule inhibitors of SEVI and other amyloid proteins.

Lipogels: single-lipid-bilayer-enclosed hydrogel spheres.

We have fabricated Lipogels consisting of a single POPC lipid bilayer supported by a micrometer-sized, thermoresponsive, hydrophobically modified (HM), hydrogel sphere. The hydrogel consists of a lightly cross-linked poly(N-isopropylacrylamide) (pNIPAM) core surrounded by a highly cross-linked acrylic acid (AA)-rich p(NIPAM-co-AA) shell. The lipid bilayer was assembled by binding liposomes to HM microgels, followed by several cycles of freeze-thaw. The pNIPAM volume phase transition (VPT) at ∼32 °C was present both before and after hydrophobic modification and after lipid bilayer coating. Fluorescence studies confirmed the fusion of liposomes into a continuous single bilayer. At a temperature above the VPT, it was found that the volume decrease in the hydrogel was coupled to the appearance of highly curved obtrusions of the uncompromised lipid bilayer into the surroundings. It is anticipated that these properties of Lipogels will prove to be useful in drug delivery applications and in fundamental biophysical studies of membranes.

From sulfur-amine solutions to metal sulfide nanocrystals: peering into the oleylamine-sulfur black box.

In this Article, we present our findings on the formation of metal sulfide nanocrystals from sulfur-alkylamine solutions. By pulsed field gradient diffusion NMR along with the standard toolbox of 1D and 2D NMR, we determined that sulfur-amine solutions used as a sulfur precursor exist as alkylammonium polysulfides at low temperatures. Upon heating to temperatures used in nanocrystal synthesis, the polysulfide ions react with excess amine to generate H(2)S, which combines with the metal precursor to form metal sulfide. Four different reaction pathways were found, each of which produced H(2)S and the byproducts identified in this Article. Thioamides were identified as an intermediate and were shown to exhibit much more rapid kinetics than sulfur-alkylamine solutions at low temperatures in the synthesis of metal sulfide nanocrystals.

The amyloidogenic SEVI precursor, PAP248-286, is highly unfolded in solution despite an underlying helical tendency.

Amyloid fibers in human semen known as SEVI (semen-derived enhancer of viral infection) dramatically increase the infectivity of HIV and other enveloped viruses, which appears to be linked to the promotion of bridging interactions and the neutralization of electrostatic repulsion between the host and the viral cell membranes. The SEVI precursor PAP(248-286) is mostly disordered when bound to detergent micelles, in contrast to the highly α-helical structures found for most amyloid proteins. To determine the origin of this difference, the structures of PAP(248-286) were solved in aqueous solution and with 30% and 50% trifluoroethanol. In solution, pulsed field gradient (PFG)-NMR and (1)H-(1)H NOESY experiments indicate that PAP(248-286) is unfolded to an unusual degree for an amyloidogenic peptide but adopts significantly helical structures in TFE solutions. The clear differences between the structures of PAP(248-286) in TFE and SDS indicate electrostatic interactions play a large role in the folding of the peptide, consistent with the slight degree of penetration of PAP(248-286) into the hydrophobic core of the micelle. This is another noticeable difference between PAP(248-286) and other amyloid peptides, which generally show penetration into at least the headgroup region of the bilayer, and may explain some of the unusual properties of SEVI.