Peter M. Macdonald

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.

Association of Highly Compact Type II Diabetes Related Islet Amyloid Polypeptide Intermediate Species at Physiological Temperature Revealed by Diffusion NMR Spectroscopy

Self-association of human islet amyloid polypeptide (hIAPP) is correlated with the development of type II diabetes by the disruption of cellular homeostasis in islet cells through the formation of membrane-active oligomers. The toxic species of hIAPP responsible for membrane damage has not been identified. In this study, we show by pulsed field gradient NMR spectroscopy that the monomeric form of the toxic, amyloidogenic human variant of IAPP (hIAPP) adopts a temperature dependent compact folded conformation that is absent in both the nontoxic and nonamyloidogenic rat variant of IAPP and absent in hIAPP at low temperatures, suggesting this compact form of monomeric hIAPP may be linked to its later aggregation and cytotoxicity. In addition to the monomeric form of hIAPP, a large oligomeric species greater than 100 nm in diameter is also present but does not trigger the nucleation-dependent aggregation of IAPP at 4 degrees C, indicating the large oligomeric species may be an off-pathway intermediate that has been predicted by kinetic models of IAPP fiber formation. Furthermore, analysis of the polydispersity of the calculated diffusion values indicates small oligomeric species of hIAPP are absent in agreement with a recent ultracentrifugation study. The absence of small oligomeric species in solution suggests the formation of small, well-defined ion channels by hIAPP may proceed by aggregation of monomeric IAPP on the membrane, rather than by the insertion of preformed structured oligomers from the solution state as has been proposed for other amyloidogenic proteins.

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.

Polylysine-Induced 2H NMR-Observable Domains in Phosphatidylserine/Phosphatidylcholine Lipid Bilayers

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.

Formation and Crosslinking of Latex Films through the Reaction of Acetoacetoxy Groups with Diamines under Ambient Conditions

We investigated the processes of film formation, polymer diffusion, and crosslinking of latex films at ambient temperature, using low Tg methacrylate latex bearing acetoacetoxy groups, and curing the systems with 1,6-hexanediamine as the crosslinker. The addition of diamine induces floc formation, which modifies the rheological properties of the dispersion and increases its drying rate when coated onto a substrate. The crosslinking reaction between diamine and acetoacetoxy groups occurs at a rapid rate, even in the dispersed state. Although the crosslinking reaction precedes polymer diffusion in the two systems we examined, latex films with relatively good solvent resistance are obtained.

Deuterium NMR studies of the interactions of polyhydroxyl compounds and of glycolipids with lipid model membranes

The physical properties of bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence of four water-soluble polyhydroxyl compounds, trehalose, sorbitol, glycerol, and ethyleneglycol, and three neutral glycolipids - monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and nonhydroxy fattyacyl-cerebrosides (NHFA-Cer) - were investigated using 2H-NMR. All four polyhydroxyl compounds induced small, but comparable concentration-dependent changes in the choline headgroup conformation which were consistent with the presence of a small negative charge being conferred upon the bilayer surface. The latter may be explained by dipolar interactions brought about by changes in the long-range order of the water layer at the membrane surface. Trehalose had a small ordering effect on the hydrophobic interior of the membrane while ethyleneglycol induced a disordering, at both the head group level and in the hydrophobic interior. The presence of high amounts of carbohydrate at the membrane surface was ensured when POPC was mixed with various proportions of one of three glycolipids, MGDG, DGDG and NHFA-Cer. In these cases the conformation of the choline headgroup was only marginally altered when not masked by macroscopic phase changes. The headgroup conformational changes observed in the presence of any of the above-mentioned compounds were modest in comparison to the effects induced by charged substances.

Surface charge response of the phosphatidylcholine head group in bilayered micelles from phosphorus and deuterium nuclear magnetic resonance

Solid-state phosphorus (31P) and deuterium (2H) nuclear magnetic resonance (NMR) spectroscopy over the temperature range of 25-50 degreesC were used to investigate bilayered micelles (bicelles) composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) in the presence of either the anionic lipid 1,2-dimyristoyl-sn-3-phosphoglycerol (DMPG) or the cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). The 31P-NMR spectra demonstrate that bicellar structures form with DMPG/DMPC ratios ranging from 0 to 50/50 and with DMTAP/DMPC ratios from 0 to 40/60, while the overall concentration of DHPC remains constant. The formation of bicelles containing charged amphiphiles is contingent upon the presence of NaCl, with 50 mM NaCl being sufficient for bicelle formation at all concentrations of charged amphiphile investigated, while 150 mM NaCl affords better resolution of the various 31P-NMR resonance signals. The 2H-NMR spectra demonstrate that the quadrupolar splittings (Deltanu) of head group-deuterated DMPC change inversely as a function of the amount of negative versus positive charge present, and that the changes for deuterons on the alpha-carbon are opposite in sense to those for deuterons on the beta-carbon. This indicates that head group-deuterated phosphatidylcholine functions as a molecular voltmeter in bicelles in much the same fashion as it does in spherical vesicles.

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.

Pulsed Field Gradient NMR Studies of Polymer Adsorption on Colloidal CdSe Quantum Dots

Pulsed field gradient nuclear magnetic resonance (PFG NMR) experiments have been used to examine ligand exchange between poly(2-(N,N-dimethylamino)ethyl methacrylate) (PDMA) (Mn = 12,000, Mw/Mn = 1.20, Nn = 78) and trioctylphosphine oxide (TOPO) bound to the surface of CdSe/TOPO quantum dots (QDs). We show that PFG 1H NMR can quantify the displacement of TOPO by PDMA through its ability to differentiate signals due to TOPO bound to the QDs versus those from TOPO molecules free in solution. For CdSe QDs with a band edge absorption maximum at 558 nm (diameter 2.7 nm by transmission electron microscopy), we determined that, at saturation, 8 polymer chains on average displace greater than 90% of the surface TOPO groups. At partial saturation, with an average of 6 polymer chains/QD, each TOPO displaced requires 28 DMA repeat units. Assuming that one Me2N- group binds to a surface Cd2+ for each TOPO displaced, we infer that only about 3% of the DMA units are directly bound to the surface. The remaining groups are present as loops or tails that protrude into the solvent and increase the hydrodynamic diameter of the particles.

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.