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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Bicellar mixtures containing pluronic F68: morphology and lateral diffusion from combined SANS and PFG NMR studies.

Small angle neutron scattering (SANS) and pulsed field gradient (PFG) nuclear magnetic resonance (NMR) diffusion measurements were applied to examine morphology and diffusion in dimyristoyl- plus dihexanoyl-phosphatidylcholine bicellar mixtures, either neutral or negatively charged, incorporating a Pluronic triblock copolymer (F68). Negatively charged bicellar mixtures, doped with dimyristoylphosphatidylglycerol (DMPG), exhibited SANS profiles consistent with a perforated lamellar morphology for the magnetically alignable phase. Correspondingly, F68 diffusion in this magnetically aligned phase was normal Gaussian, in that the mean square displacements increased linearly with the experimental diffusion time, with a lateral diffusion coefficient of 1.9 x 10(-11) m(2) s(-1) consistent with a lipid bilayer inserted configuration. Neutral bicellar mixtures, that is, lacking DMPG, in contrast, displayed SANS profiles characteristic of ribbons arranged in such a fashion as to produce extended lamellae. Within the lamellae, the ribbons exhibited an in-plane periodicity (interribbon) of between 120 and 140 A. Correspondingly, F68 diffusion was non-Gaussian, exhibiting a square root diffusion time dependence of the mean square displacement indicative of one-dimensional curvilinear diffusion. The presence or absence of DMPG, rather than of F68, dictated the ribbon versus lamellar morphology, with F68 reflecting this difference via its lateral diffusion behavior. Although ribbons have been reported previously, this is the first study to show that they aggregate, most likely into extended lamellar sheets, and eventually fold into multilamellar vesicles.

Triggered instability of liposomes bound to hydrophobically modified core-shell PNIPAM hydrogel beads.

The ability to trigger a destabilization of the membrane integrity of liposomes bound to environmentally sensitive hydrophobically modified core-shell hydrogel beads is demonstrated. Hydrogel beads with a core composed of poly(N-isopropylacrylamide) lightly cross-linked with bisacrylamide (BA) (pNIPAM) and a shell composed of NIPAM highly cross-linked with BA and containing varying amounts of acrylic acid (AA) [p(NIPAM-co-AA)] undergo a volume phase transition (VPT) at approximately 32 degrees C, as determined from (1)H magic angle spinning (MAS) NMR, regardless of the AA content of the shell. When the shell was hydrophobically modified with either decylamine or tetradecylamine, binding of extruded large unilamellar vesicles (eLUVs) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) was quantitative, as determined via fluorescence spectroscopy. Fluorescence microscopy showed that such bound eLUVs did not fuse. Hydrogel-bound eLUV membrane permeability was assessed using (31)P MAS NMR in the presence of the chemical shift agent praseodymium and demonstrated that only at lower degrees of hydrophobic modification of the core-shell hydrogels was eLUV membrane barrier integrity maintained when T < VPT. At a low degree of hydrophobic modification, cycling the temperature above the VPT even for short periods caused the eLUV membranes to become leaky. Hence, eLUV membrane permeability was coupled to the hydrogel VPT, a situation that would be useful in applications requiring triggered release of liposomal contents.

Size of bicelle defects probed via diffusion nuclear magnetic resonance of PEG.

Diffusion of various poly(ethylene glycol) (PEG) tracers of well-defined molecular weight and narrow polydispersity confined within the aqueous interstices between positively magnetically aligned bicelles was measured using pulsed-field-gradient (1)H nuclear magnetic resonance. The bicelles consisted of mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), and dihexanoylphosphatidylcholine (DHPC) in the molar ratios q = [100 DMPC +5 DMPG]/[DHPC] = 3.5, 4.5, and 5.5, to which Yb(3+) had been added in the ratio 1:75 Yb(3+)/phospholipid. The field gradients were applied such that diffusion was measured in the direction parallel to the normal to the bicelles' planar regions, thereby rendering the experiment sensitive to the ability of PEG to traverse lamellar defects within the bicelles. The pulsed-field-gradient nuclear magnetic resonance diffusive intensity decays were diffusion-time-independent in all cases, with diffusive displacements corresponding to many hundreds of bicellar lamellae. This permitted a description of such diffusive decays in terms of a mean behavior involving a combination of straight obstruction effects common to all PEG, with hindrance to diffusion proportional to the relative size of a given PEG with respect to the size of the lamellar defects. Across the range of PEG molecular weights (200-4600) and bicelle compositions examined, the apparent radial dimension of the lamellar defects decreased from 165 A with q = 3.5 to 125 A with q = 5.5. This is opposite to the trend predicted from static geometric models of either bicelle disks or perforated lamellae. Qualitatively, the observed trend suggests that mobility of the obstructions to diffusion will need to be considered to reconcile these differences.

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.

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles.

Lateral diffusion coefficients of PEG-ylated lipids with three different molecular weight PEG groups (1000, 2000 and 5000) were measured in magnetically-aligned bicelles using the stimulated echo (STE) pulsed field gradient (PEG) (1)H nuclear magnetic resonance (NMR) method. At concentrations below the PEG "mushroom-to-brush" transition, all three PEG-ylated lipids exhibited unrestricted lateral diffusion, with lateral diffusion coefficients comparable to those of corresponding non-PEG-ylated lipids and independent of PEG molecular weight. At concentrations above this transition, lateral diffusion slowed progressively with increasing concentration of PEG-ylated lipid as a result of surface crowding. As well, the lateral diffusion coefficients exhibited a pronounced decrease with increasing PEG group molecular weight and a diffusion-time dependence indicative of obstructed diffusion. We conclude that, while lateral diffusion of PEG-ylated lipids within lipid bilayers is determined primarily by the hydrophobic anchoring group, when crowding at the lipid bilayer surface becomes significant, the size of the extra-membranous domain, in this case the PEG group, can influence lateral diffusion, leading to decreased diffusivity with increasing size and producing obstructed diffusion at high crowding. These findings imply that similar considerations will pertain to lateral diffusion of membrane proteins with large extra-membranous domains.