Search

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (A β ), 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 β -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 β -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 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (A

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 co-solubilized by n-dodecyl-β-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.

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-β (Aβ)—an APP derivative and hyperphosphorylated tau form aggregates in the brain that underlie the pathogenesis of the disease. However, the connection between Aβ and tau pathologies remains unclear. Several studies have provided evidence that the presence of Aβ 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 Aβ 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.

The SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus 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 pre-formed 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 (TGN) and accumulation in swollen perinuclear LAMP1-positive vesicles, likely lysosomes. Coronavirus envelope proteins have previously been proposed to manipulate the luminal pH of the TGN, which serves as an accumulation station for progeny coronavirus particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and possibly even development of “Trojan Horse” anti-viral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

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, it seems likely that these enzymes are 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.

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.

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.

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.

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 β-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-β (Aβ) 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.

Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer´s disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether or not bexarotene induces removal of Aβ 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 Aβ 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 Aβ metabolism through multiple mechanisms.

Cationic polymers are capable of spontaneously condensing DNA into complexes suitable for nonviral gene therapy. The precisely defined molecular weights and highly symmetric surface chemistries of dendrimers, such as poly(amidoamine) (PAMAM), have made them attractive alternatives to conventional linear polymers for DNA delivery applications. In this paper, we use small-angle X-ray scattering (SAXS) coupled with osmotic stress to directly measure the effect of pH on the packaging and forces in low generation (G0 and G1) PAMAM–DNA complexes or dendriplexes. Because of the presence of both primary and tertiary amines with differing pKas, PAMAM changes its net charge with pH. We show that changing the pH at condensation results in large differences in the packaging and intermolecular forces in PAMAM dendriplexes. Both dendrimer/DNA systems show a large increase in attractions with decreasing pH scaling linearly with the inverse of the dendrimer charge, while repulsions in the system are nearly unaffected. pH...

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-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is D-galactose-α(1→6)-D-glucose. Light scattering showed the β-DDMB micelle to be roughly 30kD smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than for DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22 (PMP22). β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

DNA in vertebrate sperm nuclei is organized differently from any other cell. DNA is compacted in tightly packed (∼ 400 mg/ml) hexagonal arrays. Since DNA repair is absent in sperm, dense packaging of DNA is considered necessary to protect against damage by reactive oxidizing species. DNA damage correlates with infertility and likely contributes to miscarriages and birth defects. Our goal is to characterize protamine defects that result in DNA mispackaging. Small (∼30-50 amino acids), arginine rich (50-70%) peptides called protamines assemble DNA in sperm nuclei. Protamines will spontaneously condense DNA from dilute solution into close-packed, liquid crystalline arrays. Both sperm nuclei and reconstituted protamine-DNA arrays are sufficiently ordered that the interhelical spacing or packing density can be determined by x-ray scattering.Insufficient protamine to neutralize all DNA has been linked to excess damage. Salmon sperm nuclei can be fractionated on sucrose density gradients. The interhelical scattering maxima of the heavy and light fractions overlap indicating that all nuclei have a tightly packed fraction of DNA. The lighter fraction, however, shows a scattering profile that is skewed toward larger spacings. Part of the DNA is not as well packed as the majority. Adding excess protamine to the light fraction recovers the scattering profile of the heavy fraction.Protamines are initially serine phosphorylated when replacing histones on DNA. Incomplete dephosphorylation has also been linked to DNA damage. Herring protamines can be partially phosphorylated with protein kinase A. We find that even modest phosphorylation that reduces the protamine charge by only 5% will increase the water volume accessible to oxidizing species in reconstituted DNA-protamine assemblies by 50%.

Fetal heart rate variability has become an essential component in evaluation of the fetus during continuous electronic fetal monitoring. Because of technical deficiencies of monitoring equipment, fetal heart rate variability may not always be available, such as during indirect monitoring of the fetus. In lieu of integration, a mathematical procedure called autocorrelation has, with the use of microprocessors, been incorporated into the fetal monitor, creating the "second generation" of electronic fetal monitoring. This study examined the accuracy of the first- and second-generation monitors by comparing simultaneously obtained indirect and direct real-time fetal heart rate data. The second-generation monitor produced an indirectly obtained fetal heart rate that was more accurate when compared with the directly obtained data than that with the first-generation fetal monitor. In addition, the variation in data from the first-generation monitor was substantially higher than that with the second-generation monitor. It is concluded that the fetal heart rate tracing from the second-generation monitor is more accurate when compared with the fetal electrocardiogram and may facilitate a truer interpretation of fetal heart rate variability than that obtained with the first-generation monitor.

Metribuzin and the ethyl ester of chlorimuron were evaluated alone and in combination for preemergence broadleaf weed control in soybeans. Neither herbicide alone controlled all broadleaf weeds tested, but combinations showed both complementary and additive action. Two field studies quantified these interactions on broadleaf weeds and showed that low rates of either herbicide alone controlled Pennsylvania smartweed and redroot pigweed. Metribuzin was more effective than chlorimuron in controlling prickly sida and hemp sesbania, while chlorimuron was more effective on common cocklebur, sicklepod, and ivyleaf and pitted morningglories. Additive action was most important on velvetleaf, sicklepod, annual morningglories, and hemp sesbania. Because the components were both additive and complementary, a range of mixture rates and ratios were more effective for weed control than either herbicide alone.