Formation of extramembrane ß-strands controls dimerization of transmembrane helices in amyloid precursor protein C99.

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (Aß), is a transmembrane (TM) protein containing intrinsically disordered N- and C-terminal extramembrane domains. Using molecular dynamics (MD) simulations, we show that the structural ensemble of the C99 monomer is best described in terms of thousands of states. The C99 monomer has a propensity to form ß-strand in the C-terminal extramembrane domain, which explains the slow spin relaxation times observed in paramagnetic probe NMR experiments. Surprisingly, homodimerization of C99 not only narrows the conformational ensemble from thousands to a few states through the formation of metastable ß-strands in extramembrane domains but also stabilizes extramembrane α-helices. The extramembrane domain structure is observed to dramatically impact the homodimerization motif, resulting in the modification of TM domain conformations. Our study provides an atomic-level structural basis for communication between the extramembrane domains of the C99 protein and TM homodimer formation. This finding could serve as a general model for understanding the influence of disordered extramembrane domains on TM protein structure.

The transmembrane amyloid precursor C99 protein exhibits non-specific interaction with tau.

Historically, the two most prominent proteins in Alzheimer's disease (AD) research have been the amyloid precursor protein (APP) and the microtubule assembly protein tau. In the classical model for the etiology of AD, amyloid-ß (Aß)-an APP derivative and hyperphosphorylated tau form aggregates in the brain that underlie the pathogenesis of the disease. However, the connection between Aß and tau pathologies remains unclear. Several studies have provided evidence that the presence of Aß can induce or enhance neurofibrillary tangle formation by tau. Others have reported a direct interaction between tau and short fragments of the APP transmembrane domain, C99. Structural studies of C99 show that these in vitro tau-binding fragments of C99 are buried in the lipid bilayer and are likely unavailable to bind tau in vivo. Given the importance of APP and tau in AD, we sought to characterize the potential interaction of the Aß precursor, full length C99, and tau in vitro using NMR spectroscopy. We found that C99 and soluble tau interact only weakly and, most likely, non-specifically.

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1-positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of "Trojan horse" antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

The C99 domain of the amyloid precursor protein resides in the disordered membrane phase.

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by ß-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-ß (Aß) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid-ordered phase membrane rafts. However, direct evidence that C99 preferentially associates with these rafts has remained elusive. Here, we tested this by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles (GPMVs). We found that C99 was essentially excluded from ordered domains in vesicles from HeLa cells, undifferentiated SH-SY5Y cells, or SH-SY5Y-derived neurons; instead, ∼90% of C99 partitioned into disordered domains. The strong association of C99 with disordered domains occurred independently of its cholesterol-binding activity or homodimerization, or of the presence of the familial Alzheimer disease Arctic mutation (APP E693G). Finally, through biochemical studies we confirmed previous results, which showed that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying that either i) amyloidogenic processing of the protein occurs in disordered regions of the membrane, ii) processing involves a marginal subpopulation of C99 found in rafts, or iii) as-yet-unidentified protein-protein interactions with C99 in living cells drive this protein into membrane rafts to promote its cleavage therein.

Delivery of recombinant SARS-CoV-2 envelope protein into human cells.

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of "Trojan Horse" anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

Bicelles Rich in both Sphingolipids and Cholesterol and Their Use in Studies of Membrane Proteins.

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-ß-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.

Bexarotene Binds to the Amyloid Precursor Protein Transmembrane Domain, Alters Its α-Helical Conformation, and Inhibits γ-Secretase Nonselectively in Liposomes.

Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-ß (Aß)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aß clearance mechanism. However, whether bexarotene induces removal of Aß plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aß in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited the cleavage of Notch1, another major γ-secretase substrate, demonstrating that its inhibition of γ-secretase is not substrate specific and not mediated by acting via the cholesterol binding site of C99. Our data suggest that bexarotene is a pleiotropic molecule that interfere with Aß metabolism through multiple mechanisms.

Membrane properties that shape the evolution of membrane enzymes.

Spectacular recent progress in structural biology has led to determination of the structures of many integral membrane enzymes that catalyze reactions in which at least one substrate also is membrane bound. A pattern of results seems to be emerging in which the active site chemistry of these enzymes is usually found to be analogous to what is observed for water soluble enzymes catalyzing the same reaction types. However, in light of the chemical, structural, and physical complexity of cellular membranes plus the presence of transmembrane gradients and potentials, these enzymes may be subject to membrane-specific regulatory mechanisms that are only now beginning to be uncovered. We review the membrane-specific environmental traits that shape the evolution of membrane-embedded biocatalysts.

Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin.

Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.

Dodecyl-ß-melibioside Detergent Micelles as a Medium for Membrane Proteins.

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-ß-melibioside (ß-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the ß-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-ß-maltoside (ß-DDM). ß-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into ß-DDMB yielded activities that were 40% higher than those of DAGK purified into ß-DDM. ß-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. ß-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

Backbone Hydrogen Bond Strengths Can Vary Widely in Transmembrane Helices.

Although backbone hydrogen bonds in transmembrane (TM) helices have the potential to be very strong due to the low dielectric and low water environment of the membrane, their strength has never been assessed experimentally. Moreover, variations in hydrogen bond strength might be necessary to facilitate the TM helix breaking and bending that is often needed to satisfy functional imperatives. Here we employed equilibrium hydrogen/deuterium fractionation factors to measure backbone hydrogen bond strengths in the TM helix of the amyloid precursor protein (APP). We find an enormous range of hydrogen bond free energies, with some weaker than water-water hydrogen bonds and some over 6 kcal/mol stronger than water-water hydrogen bonds. We find that weak hydrogen bonds are at or near preferred γ-secretase cleavage sites, suggesting that the sequence of APP and possibly other cleaved TM helices may be designed, in part, to make their backbones accessible for cleavage. The finding that hydrogen bond strengths in a TM helix can vary widely has implications for membrane protein function, dynamics, evolution, and design.

Design and construction of an actively frequency-switchable RF coil for field-dependent Magnetisation Transfer Contrast MRI with fast field-cycling.

Magnetisation Transfer Contrast (MTC) is an important MR contrast-generating mechanism to characterise the MR-invisible macromolecular protons using an off-resonance pre-saturation RF irradiation pulse (or MT pulse). MTC MRI is normally implemented at a fixed magnetic field; however, it may be useful to evaluate changes of the MT effect as a function of external magnetic field strength (B0). In order to conduct field-dependent MTC experiments with a single MR system, two techniques are crucially needed. B0 should be able to be switched between levels during irradiation of the MT pulse. At the same time, the resonance frequency of the RF coil (f0) should also be able to be shifted to the corresponding value. Switching B0 is attained by the fast field-cycling technique, while in order to switch f0, a specially designed multi-tunable RF coil is required. Here, we designed and constructed an actively frequency-switchable RF coil for frequencies at and below 2.5 MHz. The design employed PIN diodes, and enabled switching f0 between five different values, with excellent impedance matching (approximately -37 dB S11 reflection) and Q-factor of about 100 at each configuration.

Cryogenic receive coil and low noise preamplifier for MRI at 0.01T.

We have investigated the design and construction of liquid nitrogen cooled surface coils made from stranded (litz) copper wire for low field MRI applications. If designed correctly, cooled litz coils can provide a competitive alternative to high temperature superconducting (HTS) coils without the complications associated with flux trapping. Litz coils can also be produced with a wider range of shapes and sizes, and at lower cost. Existing models were verified experimentally for flat spiral coils wound from solid and litz wires, operated at room temperature and 77K, and then used to design and optimise a cooled receive coil for MRI at 0.01T (425 kHz). The Q-factor reached 1022 when the coil was cooled to 77K, giving a bandwidth of just 0.42 kHz, so a low noise JFET preamplifier was developed to provide active damping of the coil resonance and thus minimise image intensity artefacts. The noise contribution of the preamplifier was determined using a method based on resistive sources and image noise analysis. The voltage and current noise were measured to be 1.25 nV/Hz(1/2) and 51 fA/Hz(1/2), respectively, and these values were used to estimate a noise figure of 0.32 dB at the resonant frequency of the cooled coil. The coil was used to acquire 0.01T spin echo images, first at room temperature and then cooled to 77K in a low noise liquid nitrogen cryostat. The measured SNR improvement on cooling, by a factor of 3.0, was found to correspond well with theoretical predictions.

Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques.

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.

Development of a 3-D, multi-nuclear continuous wave NMR imaging system.

The development of a 3-D, multi-nuclear continuous wave NMR imaging (CW-NMRI) system is described and its imaging capability is demonstrated on a range of materials exhibiting extremely short T(2) relaxation values. A variety of radiofrequency resonators were constructed and incorporated into a new gradient and field offset coil assembly, while the overall system design was modified to minimise microphonic noise which was present in an earlier prototype system. The chemically combined (27)Al in a high temperature refractory cement was imaged, and the CW-NMRI system was found to be sensitive to small differences in (27)Al content in these samples. The penetration of (23)Na in salt water into samples of ordinary Portland cement (OPC) was investigated, with enhanced uptake observed for samples with larger pore size distributions. The solid (13)C component in a carbonated cement sample was also imaged, as were the (7)Li nuclei in a sample of powdered Li(2)CO(3). A spatial resolution of 1mm was measured in an image of a rigid polymeric material exhibiting a principal T( *)(2) value of 16.3 micros. Finally, a high-resolution 3-D image of this rigid polymer is presented.

Field-cycled PEDRI imaging of free radicals with detection at 450 mT.

This paper describes the design, construction and use of a field-cycled proton-electron double-resonance imaging (FC-PEDRI) system for the detection and imaging of free radicals. The unique feature of this imager is its use of a 450-mT detection magnetic field in order to achieve good image quality and sensitivity. The detection magnetic field is provided by a superconducting magnet, giving high stability and homogeneity. Field cycling is implemented by switching on and off the current in an internal, coaxial, resistive secondary magnet that partially cancels the superconducting magnet's field at the sample; the secondary magnet is actively shielded to avoid eddy currents. EPR irradiation takes place at approximately 5 mT, following which the field is switched to 450 mT in 40 ms for NMR signal detection. Full details of the imager's subsystems are given, and experiments to image the distribution of stable free radical contrast agents in phantoms and in anesthetized rats are described.

Evaluation of MRI for in vivo monitoring of retinal damage and detachment in experimental ocular inflammation.

Two quantitative methods were developed for investigation of the potential of MRI for in vivo monitoring of retinal damage and detachment in experimental autoimmune uveitis (EAU). Measurements of retinal thickness and detachment area were performed on matched MR and histologic (HIST) images of rat eyes at different stages of EAU. In vivo MR images of rat eyes were acquired at 4.7 T using a figure-of-eight surface coil and a spin echo pulse sequence. Ex vivo measurements were performed on HIST images acquired using a digital camera attached to a microscope. MR images mirrored the HIST appearance of inflamed eyes at each stage of disease. Retinal detachments as small as 0.1 mm(2) were measured in vivo by MRI and confirmed in the same eye ex vivo by histology. Measurements performed on corresponding MR and HIST images demonstrated a good agreement between the two techniques. The potential of MRI for in vivo visualization and for monitoring changes in the eye during development of EAU was demonstrated in this study.

Continuous wave MRI of heterogeneous materials.

A prototype continuous wave MRI system operating at 7T has been used successfully to study a variety of heterogeneous materials exhibiting T2 relaxation values ranging from 10 micros to 50 ms. Two-dimensional images of a poly(methly methacrylate) (PMMA) resolution phantom (T2=38 micros) exhibited a spatial resolution of approximately 1mm at a magnetic field gradient strength of 200 mT/m. The technique was used to study the hydration, drying, and subsequent water penetration properties of cement samples made from ordinary Portland cement, and revealed inhomogeneities arising from the cure conditions. Sandstone samples from an oil reservoir in the North Sea were also studied; structure within these materials, arising from the sedimentary bed layering in the reservoir, was found to have an effect on their water transport properties. A section from a confectionery bar (T2* approximately 50-60 ms) was also imaged, and its internal structure could be clearly discerned.

In vivo detection of a pH-sensitive nitroxide in the rat stomach by low-field ESR-based techniques.

A study was made of the in vivo detectability of a pH-sensitive, imidazolidine spin probe, and the efficacy of low-frequency electron spin resonance (ESR)-based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally-detected ESR (LODESR) and field-cycled dynamic nuclear polarization (FC-DNP) for in vitro and in vivo measurements, and radiofrequency (RF)- and X-band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC-DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer-term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC-DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system.