Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2017 Oct 9;2(10):6525–6534. doi: 10.1021/acsomega.7b00619

Structural Characterization of the Amyloid Precursor Protein Transmembrane Domain and Its γ-Cleavage Site

Anna Itkin †,‡, Evgeniy S Salnikov †, Christopher Aisenbrey †, Jesus Raya †, Elise Glattard †, Vincent Raussens ‡, Jean-Marie Ruysschaert ‡, Burkhard Bechinger †,*
PMCID: PMC6645296  PMID: 31457253

Abstract

graphic file with name ao-2017-006197_0004.jpg

Alzheimer’s disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer’s disease has been associated with the amyloid β-peptide (Aβ), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. Here, we assessed the structural integrity of the transmembrane domain within oriented phosphatidylcholine lipid bilayers and determined the tilt angle distribution and dynamics of various subdomains using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopies. Although the overall secondary structure of the transmembrane domain is α-helical, pronounced conformational and topological heterogeneities were observed for the γ- and, to a lesser extent, the ζ-cleavage site, with pronounced implications for the production of Aβ and related peptides, the development of the disease, and pharmaceutical innovation.

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and is the most common form of dementia. The statistics for 2015 states that nearly 47 million people worldwide have AD or a related dementia. The disease is characterized by brain cell destruction, memory loss, and deterioration of cognitive and behavioral processes, severe enough to affect work, lifelong hobbies, and social life. Symptoms worsen over time, and the disease is always fatal. Small soluble or protofibrillar assemblies of amyloid β peptide (Aβ) are believed to be involved in the onset and progression of AD.1−4 The length of Aβ peptides varies from 38 to 43 residues, with Aβ40 and Aβ42 being the major species.5 All Aβ peptides are products of a proteolytic cleavage of the amyloid precursor protein (APP), an integral membrane protein with a single transmembrane domain (TMD), expressed in many tissues and concentrated in the synapses of neurons.6−9 Its primary function is not known, although cell-associated APP has been shown to be involved in some aspects of neuronal development, signalling, intracellular transport, and neuronal hemostasis.10 Nevertheless, it is most commonly studied in connection with Aβ peptides′ production.6−8,11

Notably, the resulting cleavage products and their oligomeric fiber intermediates have been suggested to be involved in memory loss via their cell cytotoxity. Because membranes assist in the formation of such toxic species, the bilayer interactions are considered of utmost importance for the development of AD and need further investigation.12,13

Amyloidogenic processing of APP begins with proteolytic cleavage of the bulk ectodomain by membrane-bound β-secretase, leading to a secreted fragment sAPPβ and a membrane-bound β-C-terminal fragment (β-CTF), also known as C99.8,14 Following transmembrane proteolysis of β-CTF by γ-secretase at the ε- and γ-sites (Figure 1) results in Aβ production and release of the APP intracellular domain.8 Accumulated evidence suggests that Aβ peptides are released via a sequential proteolytic cleavage mechanism, where the first cut occurs at residue 720 or 719 of β-CTF, that is, at the ε-site located at the cytoplasmic edge of the TMD (Figure 1).15 Products Aβ49/Aβ48 remain membrane-bound and are further processed in a sequential mode through the ζ-site (residue 717 of APP) to yield Aβ46.16 Aβ46 is the precursor of Aβ40 and Aβ42, processed via γ-cleavage.17 The Aβ production in the β-secretory pathway is promoted by APP homodimer formation,18 and further cleavage that generates e.g., Aβ38 and Aβ37 will occur only if not inhibited by strong dimerization between two TMD helices.19,20

Figure 1.

Figure 1

Open in a new tab

Partial sequence of APP, numbered following the full-length protein sequence. The TMD region in LMPG micelles from amino acid 700 to 723 is underlined;21 in bilayers, the helix ends at L720/V721.25 The shaded sequence marks APP_TM4K, the region synthesized for this study. Underlined with red are isotopically labeled amino acids that were incorporated for solid-state NMR studies. Positions of the glycines in three consecutive GxxxG sequences are shown in bold and numbered according to the APP sequence (i.e., 696, 700, 704, 708). Cleavage sites of α-, β-, and γ-secretases are indicated by triangles. γ-secretase cleavage sites correspond among others to amino acids 709, 710, 711, and 713 and upon cleavage by secretases produce Aβ38, Aβ39, Aβ40, and Aβ42, respectively.

Multidimensional solution NMR spectroscopy in micellar environments indicates that the TMD of APP is a curved helix with its apex close to G708 and G709, that is, in the center of the hydrophobic region, which results in a 17 ± 7° change in helix alignment.21,22 However, MD calculations show that this kinked structure is specific for micellar but not bilayer environments.23 At its amide terminus, the TMD is connected by a loop to another less-stable helical domain in the juxtamembrane region oriented along the lysomyristoylphosphatidylglycerol (LMPG) micellar surface.21 When investigated at peptide-to-dodecylphosphocholine (DPC) ratios of 1/50, a mixture of monomer and dimer occurs with closely related structures.22,23 The dimer interaction site involves the extended heptad repeat I702–L723 encompassing the γ-secretase cleavage sites.22

In view of the dynamic nature of the APP TMD and the influence of membrane curvature on structural details, biophysical measurements in lipid bilayers are sought after.23 Indeed, magic angle spinning NMR chemical shift measurements indicate that in lipid bilayers helical and nonhelical conformations of individual residues within the TMD are in slow exchange,24 conformational equilibria that are sensitive to lipid composition and temperature. Whereas helical structures are favored by cryotemperatures and/or PG lipids, the amount of nonhelical conformations at residues around the γ-cleavage site increases in membranes mimicking more closely the brain lipid composition and at room temperature.24,25 The helical TMD ends around residues L720/V721, which positions the ε cleavage in a less-structured region.25

In lipid bilayers, solid-state NMR intermolecular cross-peaks suggest that the APP TMD tends to assemble into a right-handed coiled-coil dimer,25 in agreement with recent MD calculations.26 The interaction surface of the homodimer includes G700, G704, and G708, but not G709 and A713.,25a Indeed, such GxxxG motifs are known to mediate transmembrane helix dimerization,27 and mutational studies have confirmed the importance of these glycines for APP processing.28−30 Furthermore, accumulated structural and biochemical data suggest that the conformational variability of the TMD segment plays an important role in APP enzymatic cleavage and therefore in Aβ production.24,26,28,31,32 Thereby, intramembraneous proteolysis by γ-secretase probably requires local unraveling of the helical secondary structure of the TMD to expose the backbone carbonyl carbons for nucleophilic attack by polarized water in the enzyme active site.

Importantly, whereas a number of different conformers are observed during MD calculations, the type of conformer is heavily dependent on the macroscopic shape and curvature of the membrane model system,31 where the right-handed coiled-coil dimer is dominant in 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers26 but a left-handed parallel dimer predominates in dodecylphosphocholine micelles.22 Important structural details such as the helical outlines, the overall molecular shape, or the topology of the dimer differ between micelle and lipid bilayer environments.21−25 Therefore, these previous investigations underline the importance of structural investigations in liquid crystalline bilayers. Here, we used solid-state NMR spectroscopy of supported planar lipid bilayers to study conformational details of the TM homodimer, where the same peptide used in a previous NMR investigation was studied by highly complementary approaches.24 The investigations are all performed in liquid crystalline bilayers, which imposes additional challenges due to the fluidity of the lipids and the dynamics of the embedded peptides. Solid-state NMR spectroscopy is unique in providing accurate structural information under such near-physiological conditions. This is particularly important when a lipid bilayer environment is required to reveal precise structural details of APP but also because its cleavage products aggregate in a lipid-dependent manner.12,13 Notably, these are the first solid-state NMR investigations of the APP TMD reconstituted in supported lipid bilayers that are oriented relative to the magnetic field of the NMR spectrometer. In particular, the data reveal a structuration into subdomains, dynamic features of individual sites and the resulting membrane topologies, and orientational distribution and conformational heterogeneity of the TMD.

Results

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) Spectroscopy

To investigate the global secondary structure of the APP_TM4K peptides reconstituted into POPC bilayers and the alignment of lipids and peptides in the sample and to assess the overall peptide orientation (i.e., transmembrane or in-plane), ATR-FTIR spectroscopy was used. When recorded at ambient temperatures, the amide I region of the spectrum (1700–1600 cm–1) exhibited a symmetric peak with a maximum at 1653 cm–1, characteristic of predominantly helical conformations (Figure 2). Fourier self-deconvolution revealed several components within the amide I region, which were quantified by an iterative least-squares curve-fitting procedure.33 The calculated secondary structure content was found to be about 74% for the helical structure and 18% for random coil and turn conformations. The remaining 8% are within the experimental error.

Figure 2.

Figure 2

Open in a new tab

ATR-FTIR spectra of 2 mol % APP_TM4K reconstituted into oriented POPC bilayers. The red spectrum was obtained with 90°-polarized light, whereas the green spectrum was obtained with 0°-polarized light. The calculated dichroic spectrum (black spectrum) is the difference between the red and green spectra. Spectra are recorded at ambient temperature and have been shifted for better visualization.

The amide I region of the spectra was also used to determine the orientation of the α-helical APP_TM4K peptide relative to the POPC hydrocarbon chains of the bilayer. A dichroic spectrum of the amide I band is strongly 90° polarized, as seen from the positive deviation in the helical region (Figure 2, black trace). This indicates that the α-helix orientation is perpendicular to the germanium plate and therefore parallel to the acyl chains of the lipids. A quantitative estimation of the helix orientation was obtained by computing an isotropic dichroic ratio of 1.16 using the lipids′ ν(C=O) band at 1738 cm–1 as a reference.34 With this calibration, the dichroic ratio of the amide I band was 1.74. When only the helical component of the amide I band was analyzed, the tilt angle relative to the normal of the germanium plate was found to be ≤20°. The orientation of the lipid acyl chains was also assessed. The lipid dichroic ratio for the band at 2872 cm–1 (νs (CH3)) was found to be 3.71 (data not shown), and accordingly, the tilt angle between acyl chains and the normal of the germanium crystal was ≤25°. This result indicates that APP_TM4K reconstituted into POPC bilayers forms a predominantly transmembrane α-helix.

15N and 2H Solid-State NMR Spectroscopy: APP_TM4K Orientation and Conformation

Local conformational details of the membrane-inserted APP_TM4K helix were revealed by preparing peptides selectively labeled with stable 15N and 2H isotopes, followed by reconstitution into uniaxially oriented POPC bilayers and solid-state NMR investigations at 296 K (Figure 3A–E). In the presence of POPC, dynamic nonhelical conformers are favored at some positions within the overall helical transmembrane region, similar to the observations made in brain lipid extracts and membrane mimetics.24 These are thought to be of particular importance for cleavage by secretases.19,24,25 Peptides with three different labeling schemes were used: 15N-Gly708/2H-Ala713, 15N-Ala713/2H-Ala701, and 15N-Val717/2H-Ala701.

Figure 3.

Figure 3

Open in a new tab

Proton-decoupled 15N (A–C) and 2H solid-state NMR spectra (D, E) of 2 mol % APP_TM4K peptides reconstituted into oriented POPC lipid bilayers. The labeled sites are 15N-Gly708 (A), 15N-Ala713 (B), 15N-Val717 (C), 2H-Ala701 (D), and 2H-Ala713 (E). (F) Representative proton-decoupled 31P spectrum of a sample. All spectra were obtained with the membrane normal oriented parallel to the magnetic field direction. In panels (D) and (E), the central small splitting of ≤1.5 kHz is from residual deuterium of the membrane hydration shell.62 The spectra were recorded at ambient temperature. The smooth lines overlaying the spectra are spectral simulations with the following parameters: the angle between the main axis of the α-helix and membrane normal were (A) 23°, peak maximum at 192 ppm; (B) 70°, peak maximum at 85 ppm; and (C) 15°, peak maximum at 210 ppm, or the Cα–Cβ bond angles were 49 and 52°, respectively (D and E). The Gaussian distributions for the peptide alignment are 3° (A), 35° (B), 20° (C), and 10° (D, E). For the simulation of the 31P spectrum (F), the Gaussian distribution of 3° was summed up with 25% powder pattern. A spectral line width of 500 Hz (A–C, F) and 2 kHz (D, E) was applied.

On the one hand, the proton-decoupled 15N solid-state NMR spectra of the 15N-labeled peptide amide bonds provide a direct indicator of the approximate tilt angle of the 15N–1H-vector and thereby also of helical domains.35 Whereas chemical shifts in the 200 ppm region are associated with transmembrane helices, on the opposite, values <100 ppm are indicative of in-planar orientations. On the other hand, the 2H quadrupolar splitting of methyl-deuterated alanine provides highly complementary information on the orientation of the Cα–Cβ2H3 vector, which can be used to refine the helical tilt and pitch angles.36,37 Finally, proton-decoupled 31P solid-state NMR spectra were recorded for each sample to assess the quality and the alignment of the lipids. The POPC lipids predominantly resonate at 30 ppm, typical of phosphatidylcholine molecules in liquid-disordered bilayers that are oriented with their normal parallel to the magnetic field direction and thereby the bilayer normal.36,37 In the presence of polypeptides that disrupt the membrane packing, the 31P solid-state NMR spectra often extend up to −15 ppm that arise from different lipid alignments.38,39 A representative spectrum is shown in Figure 3F and spectra from other samples in the Supporting Information (Figure S1).

Figure 3A shows the 15N-Gly708 spectrum with a chemical shift value at 191 ppm, which indicates that Gly708 is in a transmembrane orientation.35 The line width at half-height is <12 ppm, demonstrating that the peptide is well-oriented with respect to the magnetic field direction. The 2H spectrum of Ala713 recorded on the same sample did not exhibit one clearly distinguishable quadrupolar splitting, but rather a distribution of Cα–Cβ2H3 alignments that add up to the shape seen in Figure 3E. The width of the spectrum is ∼20 kHz, which can be simulated by Cα–Cβ2H3 alignments relative to the membrane normal (magnetic field direction) at an average angle of 52° and a Gaussian distribution with 10° standard deviation (Figure 3E).

When this same site was labeled with 15N, the corresponding 15N chemical shift solid-state NMR spectrum of Ala713 exhibited a complex spectral line shape, where the 15N chemical shift values were spread over 160 ppm (Figure 3B), that is, covering almost the full chemical shift anisotropy, indicating that the 1H–15N vector of the Ala713 site is characterized by a wide range of orientations relative to the membrane normal, due to structural and/or orientational heterogeneity of the Ala713 site. Notably, the 2H solid-state NMR spectrum recorded from the 2H3-Ala701 label of the same sample did not resemble a powder pattern line shape (Figure 3D), as would have been expected if the peptide or the sample as a whole was misaligned. In addition, the corresponding proton-decoupled 31P solid-state NMR spectrum exhibited a line shape typical of well-aligned POPC lipids (Figure S1C). Consequently, the combination of 15N and 2H solid-state NMR spectra of Ala713 (Figure 3B,E) shows that this site adopts a range of different orientations, which exchange only slowly on the NMR time scale (kHz range).

When the peptide carrying the 15N-Val717 site is reconstituted into uniaxially oriented membranes, 15N chemical shift intensities are observed at 209 ± 28 ppm (Figure 3C). Although a broad spectral resonance with a correspondingly higher noise level is observed, the chemical shift values undoubtedly indicate a transmembrane orientation of the Val717 segment.35 The width of the spectrum points toward a distribution of orientations around a main value of the Val717 15N–1H vector relative to the magnetic field direction. Whereas the mean value of 209 ppm is indicative of a helical tilt angle around 15° (5–25° depending on the pitch angle), a rather large range of possible helix/15N–1H vector alignments from 0 to 50° results from taking into account the full spectral width (Figure 3C).

The same peptide carrying the 15N-Val717 label was also labeled at 2H-Ala701, and the corresponding 2H spectrum exhibits a maximal quadrupolar splitting of 26 kHz (Figure 3D). The width of the peptide spectrum and its shape point toward a heterogeneous distribution of alignments also for Ala701.

Discussion

Multidimensional solution-,21,22 MAS-,24,25 and oriented solid-state NMR spectroscopies, as well as MD simulations,23 all agree that in membrane environments the TMD of APP exhibits predominantly helical conformations. The helix extends from residues G700–L723 in LMPG micelles21 and from K699–K724 in the presence of DPC22 and ends around residues L720/V721 in lipid bilayers, that is, the position of the ε-cleavage site.25 ATR FTIR spectra indicate a global tilt angle of the helical part of approximately 20° (Figure 2). Furthermore, for several amino acid positions along the TMD, equilibria between helical and nonhelical conformers were detected by MAS solid-state NMR spectroscopy.24,25 Whereas helical conformations are favored by cryotemperatures and/or the presence of PG lipids,25 the amount of nonhelical conformations at residues close to the amino- and carboxy-termini of the helix and around the γ-cleavage site increases in POPC, brain lipid extracts, or model membranes mimicking the brain lipid composition and at higher temperatures.24 Here, such previous investigations of conformational equilibria were considerably extended and refined not only by identifying nonhelical residues but also by investigating the structure, dynamics, and topological distribution of the resulting transmembrane subdomains. The supported POPC lipid bilayers used here were selected to obtain new and more detailed structural insight into physiological temperatures and under conditions emphasizing the nonhelical structures also observed in brain lipids.

Using solid-state NMR on planar supported lipid bilayers, orientational restraints have been obtained from various sites along the TMD of APP labeled selectively with 2H3-alanine and/or 15N at the corresponding peptide bond. In contrast to MAS solid-state NMR, the technique exploits the full range of the chemical shift anisotropy and thereby provides information, at atomic resolution if a site is part of a well-defined secondary structure, on motions that cause dynamic averaging as well as structural information in terms of angles and their distribution. Importantly, in the experiments designed here both the amide 15N and the 2H3-alaninine labels reveal details about the alignment and the alignment distribution of the peptide backbone relative to the magnetic field of the NMR spectrometer (Bo), which is collinear with the sample normal. The 31P solid-state NMR spectra indicate well-oriented samples, where all of them exhibit closely related 31P NMR line shapes (Figures 3F and S1). The pronounced differences of 2H and 15N spectra from various labeled sites of the peptides (Figure 3A–E) thus represent conformational and topological differences of the polypeptide, rather than artifacts of individual preparations.

The 15N solid-state NMR spectra of Gly708 and Val717 are indicative of structured helical domains with transmembrane topology (Figure 4), thereby extending previous NMR and MD structural data.21−25 Notably, the Gly708 site represents the apex of the bent helical conformation, which is observed only in micellar environments.21−23 Whereas the simulations in Figure 3 compare the spectra that result from a specific alignment to those from the experimental result, Figure 4 indicates all combinations of tilt and pitch angles that agree with a 15N spectrum. On the other hand, the 2H spectra are a direct indicator of Cα–Cβ orientations relative to magnetic field direction Bo (coincident with the membrane normal) and highly sensitive to its mosaicity.37

Figure 4.

Figure 4

Open in a new tab

Contour plots illustrating the helical topologies of the APP_TM4K peptide, which agree with the experimental measurements of the 15N chemical shift of (A) (192 ± 7) ppm of Gly708 and (B) (209 ± 28) ppm of Val717. The x-axis shows the rotational pitch angle around the helix long axis, whereas the y-axis shows the tilt angle, relative to the membrane normal. The gray area represents the possible pitch/tilt angular pairs that agree with the most intense chemical shift value (bold line) and the chemical shifts corresponding to the intensities at half-height, where the intervals of the contours are in steps of 7 ppm.

Furthermore, the effects of wobbling and rocking motions of the helices on the tilt and rotational pitch angles are estimated in Figures S2 and S3. These spectral simulations indicate that taking into consideration typical motions of transmembrane sequences40,41 shifts the fits to a more transmembrane orientation and larger fluctuations of the Cα–Cβ bonds. Both change by about 5–10°, thereby also providing an estimate of error bars that arise from uncertainties about the exact motional regime.

In an oriented POPC lipid bilayer and at ambient temperatures, the A713 15N solid-state spectrum shows a broad resonance with a maximum at 85 ppm extending to about 200 ppm (Figure 3B). The spectral line shape is indicative of a preference for angles close to perpendicular to the membrane normal albeit with additional alignments of the N–H vector covering almost all possible directions. A spectral simulation with a helix tilt angle of 70° and a Gaussian distribution of 35° reproduces the experimental spectrum reasonably well (Figure 3B). Therefore, the broad, nonaveraged 15N spectral line shape (Figure 3B) refines previous MAS NMR measurements, which had been rationalized by exchange between helical and nonhelical conformations that is slow on the millisecond time scale.24 Despite the broad distribution of the 15N–1H vector, the 2H-solid-state NMR spectrum from the deuterated methyl group of the same amino acid agrees with a comparatively well-defined tilt angle of the Cα–Cβ bond of 52 ± 10° (Figure 3E). The combination of both the 15N and 2H measurements indicates that the relative orientation of the Cα–Cβ bond undergoes only modest variation relative to the sample normal, when at the same time, it suggests that rotation around the Cα–Cβ bond scans a wide variety of N–H alignments (Figure 5). Additional variability of the NH bond topology is obtained through motions involving the Ramachandran angles. An alternative analysis of the topological restraints of the A713 2H data is presented in Figure S4, together with a spectral simulation of the corresponding 15N spectrum. Both approaches indicate that the transmembrane helical conformation is interrupted at this site. Notably, residue 713 is next to the γ-secretase cleavage site V711/I712 and the break in helical conformation may be important for enzymatic activity (Figure 1). Our data thus nicely consolidate previous MAS solid-state NMR investigations of I712 and A713 at higher temperatures, which suggested mixed conformations in this region but suffered from low signal-to-noise ratios.24,25

Figure 5.

Figure 5

Open in a new tab

Model of the Ala713 site. (A) Oriented 2H solid-state NMR spectra (Figure 3E) are indicative of an alignment of the Cα–Cβ vector (black line and cone) at 52 ± 10° relative to the magnetic field direction (Bo, arrows pointing upward). The predominant 15N chemical shift intensity agrees with a 15N–H vector alignment of ca. 70 ± 35° (or because of symmetry 110 ± 35°, blue line). (B) The broad line shape of the 15N spectrum with smaller intensities reaching into the 200 ppm region indicates that NH alignments parallel to Bo also occur (Figure 3B). Indeed, rotation around the Cα–Cβ bond results in a range of NH orientations from nearly parallel to Bo to 110°. The carbons are black, nitrogen is blue, oxygen is red, and hydrogen is white. Additional degrees of freedom occur around two Ramachandran angles Φ, Ψ (not shown).

The V717 site represents the most C-terminal residue investigated in this article (Figure 3C). The spectrum is indicative of a transmembrane helical arrangement albeit with considerable topological and/or conformational dispersion. Interestingly, this site is part of the V717/I718 ζ-cleavage site (Figure 1).

Finally, the 2H solid-state NMR spectrum of A701 (Figure 3D) agrees with a Cα–Cβ bond orientation of 49 ± 10°. However, additional contributions are needed to fully account for the spectral features. The predominant 2H quadrupolar splitting of ∼27 kHz indicates that motions are slow or intermediate on the corresponding time scale (Figure 3D,E). Indeed, in the same lipid bilayer used here, two sets of Cα, Cβ and CO chemical shifts were observed in 13C MAS solid-state NMR spectra, indicative of helical and nonhelical conformations in slow exchange.24 In micellar environments, position 701 is within the first helical turn of the TMD.21,22 Notably, the residue is also within a domain that has been identified to be part of the cholesterol binding site,21 a lipid important for γ-secretase activity and raft formation.42,43 The dynamic nature of the APP TMD contrasts with that of other membrane-associated polypeptide sequences for which it was possible to determine a unique topology/tilt angle for the helical domain.41,44,45

Previous investigations show that the same transmembrane sequence investigated here adopts a right-handed coiled-coil dimer arrangement in POPC bilayers,25 in excellent agreement with studies in the presence of detergent micelles at similar peptide-to-amphiphile ratios.22,46 Familial Alzheimer mutants of APP have been found to increase the population of monomers46 when at the same time increased dimerization results in decreased Aβ production.29,47 Notably, only small differences in chemical shift were observed in previous investigations when the mono- and dimer were compared with each other in micellar environments, suggesting only small conformational changes upon dimer formation.22

A model summarizing our structural data is shown in Figure 6. Whereas residue 701 is relatively mobile being close to the amino-terminal end of the transmembrane helix, residue 708 is well-structured and exhibits a unique and well-defined topology in POPC planar lipid bilayers. Notably, 708 is also part of the helical domain involved in homodimer formation in lipid bilayer where the interaction surface involves G700, G704, and G708.25 Residue 708 is also involved in dimerization when the APP TMD is studied in DPC micellar environments,22 albeit the handedness of the dimer arrangement depends on the membrane curvature,23 being left-handed in micelles22 and right-handed in bilayer environments.25 On the one hand, the bilayer topologies measured here (Figure 4) agree with coiled-coil helical crossing angles ≤60° observed in DPC micelles22 or previously established models that were used to analyze measurements in phospholipid bilayers.25 On the other hand, in our study, both the amino- and carboxy-terminal helices adopt transmembrane alignments, whereas a pronounced kink between these helices and consecutively a more in-planar alignment of the N-terminal domain have been suggested for the monomeric protein in detergent micelles.21

Figure 6.

Figure 6

Open in a new tab

Model summarizing our structural data of the TMD dimer, including sites of structural variability/flexibility.

Indeed, not only the conformation of the cleavage site itself but also the overall shape of the transmembrane domain has been suggested to be important for interactions with the secretases.21 The oriented solid-state NMR data presented here (Figures 3 and 5) show a conformational equilibrium, which interrupts the helix around position 713 and provides flexibility for increased topological heterogeneity of its carboxy-terminal domain including residue 717. The latter is characterized by a 15N–H vector being aligned in a transmembrane fashion, albeit covering a considerable angular distribution. In addition to the A713 residue (Figure 3B,E), the G708/G709 site has also been identified as a site with high flexibility.19,21,22 Such hinge regions within the helical domain probably help the TMD to fit into the secretase binding site. At the same time, pronounced conformational and orientational heterogeneity of the γ- and, to a smaller extent, the ζ-cleavage site (Figure 3B,C) may have important implications for the cleavage mechanism of APP and hence for Aβ production and can serve as sites of enzymatic activity.

This view of a rather heterogeneous structural arrangement, enriched by detailed information on the topology of 15N–H and Cα–Cβ2H3 directors, much refines MAS solid-state NMR investigations, which showed the coexistence of nonhelical and helical conformations24 as well as MD simulations, which exhibit a dynamic exchange of conformers where the conformational equilibria are shifted by single-residue mutagenesis19 or lipid composition.26 Additional structural and topological details could be obtained from two-dimensional solid-state NMR spectra of uniformly 15N-labelled APP reconstituted into oriented phospholipid bilayers48 although the dynamic nature of some of the sites will probably make their investigation, together with many other residues, more difficult. The dimerization and structural transitions, which are regulated by the lipid composition24 and membrane curvature,31 may be coupled to each other and important for regulation of availability and access of the substrate to the γ-secretase enzymatic site. Notably, in recent electron microscopy structural investigations, single transmembrane domains of γ-secretase also appear quite dynamic49 and such regulatory mechanisms may be enhanced by the enzyme activity itself, being dependent on the membrane lipid composition.42,43,47

Experimental Section

APP_TM4K Peptide Synthesis

The 30 amino-acid-long polypeptides, which correspond to the transmembrane part of full-length APP (amino acids 699–726, KGAIIGLMVGGVVIATVIVITLVMLKKK), were synthesized by solid-phase peptide synthesis using Fmoc chemistry50 on a Millipore 9050 automated peptide synthesizer (Millipore, Darmstadt, Germany) using a TentaGel R RAM resin (polyethylene glycol polystyrene-graft copolymer support) with a degree of substitution of 0.190 meq/g (Rapp Polymere GmbH, Tübingen, Germany). The synthesis was carried out at 40 °C, with double couplings for Val715 and Ile716 and acetylation of nonreacted peptides. Side-chain protecting groups and peptide cleavage from the resin was carried out at room temperature for 4 h in 94% aqueous trifluoroacetic acid (TFA) with the addition of 2.5% ethanedithiol, 1% of the silane derivative triisopropylsilan and 2.5% water as scavenger cocktail. The yield of the crude peptide varied from 60 to 90%.

Peptides were purified by high-performance liquid chromatography (HPLC) using a reversed-phase Prontosyl 300-5-C4 (particles size of 5 mm with 300 Å pores) column (Bishoff, Leonberg, Germany). The mobile phase consisted of a gradient of isopropanol with 0.1% TFA (buffer B) mixed with water and 0.1% TFA (buffer A). The content of buffer B increased from 10 to 60% in 23 min at a flow rate of 10 mL/min. All of the solvents were of highest purity (HPLC grade, Sigma-Aldrich Chimie S.a.r.l., Lyon, France). The quality of the purified peptides was assessed by matrix-assisted laser desorption/ionization (MALDI) with a time-of-flight (TOF) mass spectrometer (Autoflex from Bruker Daltonics, Bremen, Germany). The yield of the pure peptide varied between 10 and 30%. Purified APP_TM4K peptides were lyophilized 3 times from 10 mM HCl to remove the TFA counterions.51 In the presence of TFA, FTIR spectra show a sharp and intense peak at 1675–1680 cm–1. Such an intensity is absent in the experimental spectra, indicating that the TFA ions have been successfully removed, as demonstrated previously.51

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR)

ATR-FTIR spectra were recorded on a Bruker IFS 55 infrared spectrophotometer (Ettlingen, Germany) equipped with a liquid-nitrogen-cooled MCT detector. The internal reflection element (IRE) was a germanium (Ge) ATR plate (50 × 20 × 2 mm3) with an aperture angle of 45°, yielding 25 internal reflections. A total of 128 scans were accumulated for each spectrum to improve the signal-to-noise ratio. Spectra were recorded at a nominal resolution of 2 cm–1. The spectrophotometer was continuously purged with dried air. All of the measurements were carried out at 24 °C.

APP_TM4K peptides were dissolved in HFIP (Sigma-Aldrich Chimie S.a.r.l., Lyon, France) to a final concentration of 2 mg/mL. This solution was mixed with POPC lipids (Avanti Polar Lipids, Alabaster, AL) dissolved in chloroform (Sigma-Aldrich Chimie S.a.r.l., Lyon, France) to obtain a 1:50 peptide-to-lipid ratio. The solvents were evaporated, and the sample was spread on the surface of the Ge plate to form a thin film of oriented multilayers.

Secondary Structure Determination

Fourier self-deconvolution was applied to increase the resolution of the spectra in the amide I region, which is highly sensitive to the protein secondary structure. Lorentzian line shapes were used for self-deconvolution and a Gaussian line shape for apodization. A least-squares iterative fitting of the spectrum between 1700 and 1600 cm–1 using a mixture of Gaussian/Lorentzian line shapes was performed, and the area belonging to different secondary structures was determined. Prior to curve fitting, a straight base line passing through the ordinates at 1700 and 1600 cm–1 was subtracted. To avoid the introduction of artifacts, the fitting was done on the nondeconvoluted spectrum. The proportion of a particular structural element was computed to be the sum of the area of all of the fitted bands with their maximum in the frequency region concomitant with that particular structure divided by the area of all of the Lorentzian bands having their maximum between 1689 and 1615 cm–1.33

Orientation of the Secondary Structure

The thickness of the deposited film was considered to be small compared with the IR wavelength, and the “thin film” approximation was used when establishing the equations describing the dichroic ratio as a function of the orientational order parameter.33 Spectra were recorded with the incident light polarized either parallel (0°) or perpendicular (90°) relative to the plane of the internal reflection element (IRE), that is, the Ge plate. Polarization was expressed as dichroic ratio, RATR = A90/A0°, and the mean angle between the helix axes and a normal to the ATR plate surface was calculated. In these calculations, an angle of 27° between the long axis of the α-helix and the C=O dipole moment was taken into account.33,52,53 The POPC ester stretch ν(C=O) vibrations at 1738 cm–1 were used to characterize the isotropic dichroic ratio.

Solid-State NMR Spectroscopy

The following isotopic labeling schemes were introduced into the APP_TM4K peptides during solid-phase synthesis: 15N-Gly708/2H-Ala713, 15N-Ala713/2H-Ala701, and 15N-Val717/2H-Ala701. The oriented samples were prepared as described in detail in ref (54). A peptide-to-lipid ratio of 1:50 was obtained by mixing 3.5 mg of dry peptide dissolved in HFIP (Sigma-Aldrich Chimie S.a.r.l., Lyon, France) with 46.4 mg of POPC dissolved in chloroform. Before NMR measurements, the samples were equilibrated at 93% relative humidity at room temperature. For 2H experiments, the saturated solution was prepared with deuterium-depleted water with a residual degree of deuteration of 10–6. During the solid-state NMR experiments, the samples were cooled by a stream of air at 296 K.

Proton-decoupled 31P solid-state NMR spectra were recorded at 202.41 MHz on a Bruker Avance wide-bore NMR spectrometer operating at 11.7 T (Bruker, Rheinstetten, Germany). A Hahn echo pulse sequence55 was used with the typical parameters: 1H B1 field 33 kHz, 90° pulse length 10 μs, echo delay 100 μs, spectral width 100 kHz, 2048 data points, typically 512 scans, and repetition time 3 s. The spectra were referenced relative to 85% phosphoric acid (0 ppm). An exponential apodization function corresponding to a line broadening of 100 Hz was applied before Fourier transformation.

Proton-decoupled 15N cross-polarization (CP) spectra of static aligned samples were acquired at 50.67 MHz on a Bruker Avance wide-bore NMR spectrometer operating at 11.7 T (Bruker, Rheinstetten, Germany). A commercial E-free triple-resonance solid-state NMR probe equipped with a flat coil56 was used (Bruker, Rheinstetten, Germany). The adiabatic passage through the Hartman-Hahn CP condition (APHH-CP) pulse sequence57 was used with the typical parameters: spectral width 35.7 kHz, acquisition time 14.4 ms, and CP contact time 600 μs. The 1H B1 field was swept from 40 to 60% following a tangent amplitude-modulated slope, and the average value corresponded to 47.6 kHz. About 60 000 scans were accumulated with 1024 points. An exponential apodization function corresponding to a line broadening of 300 Hz was applied before Fourier transformation. Spectra were externally referenced to 15NH4Cl at 41 ppm.58

2H solid-state NMR spectra were recorded using a quadrupolar echo pulse sequence59 with the typical parameters: 2H B1 field 40.3 kHz, echo delay 40 μs, spectral width 500 kHz, 16 000 data points, about 120 000 scans, and repetition time 1.5 s. The spectra were referenced relative to 2H2O (0 ppm). The FID was transformed beginning at the exact top of the echo. An exponential apodization function corresponding to a line broadening of 100 Hz was applied before Fourier transformation.

Calculation of Orientational Constrains Obtained from ssNMR Data

To evaluate APP_TM4K orientations that agree with the experimental results, the static 15N chemical shift tensor was oriented with respect to the molecular coordinate system of the polypeptide structure using an Euler algorithm as described before.3615N chemical shift main tensor elements of 56, 81, and 223 ppm for alanine and valine and 40, 67, and 217 ppm for glycine, respectively, were used.60 To evaluate the peptide orientations that agree with the experimental results of the 2H quadrupole interactions of the alanine residue, a maximal quadrupolar coupling constant of 74 kHz was used for the alanine 2H3C-group.61

Acknowledgments

This research project has been supported by the European Commission under the 6th Framework Programme through the Marie-Curie Action: BIOCONTROL, contract number MCRTN – 33439. The financial contributions of the Agence Nationale de la Recherche (projects TRANSPEP, ProLipIn, membraneDNP, InMembrane, and the LabEx Chemistry of Complex Systems), the University of Strasbourg, the CNRS, the Région Alsace, and the RTRA International Center of Frontier Research in Chemistry are gratefully acknowledged. V.R. acknowledges funding from the F.R.S-FNRS (PDR #70214.12) and the SAO-FRA (grant S#14025). V.R. is a Senior Research Associate for the National Fund for Scientific Research (F.R.S-FNRS, Belgium).

Glossary

Abbreviations

AD

Alzheimer’s disease

APP

amyloid precursor protein

ATR FTIR

attenuated total reflection Fourier transform infrared

β-CTF

β-C-terminal fragment

DPC

dodecylphosphocholine

EPR

electron paramagnetic resonance

HFIP

1,1,1,3,3,3-hexafluoro-2-propanol

LMPG

lysomyristoylphosphatidylglycerol

MAS

magic angle spinning

MD

molecular dynamics

NMR

nuclear magnetic resonance

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

SDS

sodium dodecyl sulfate

TMD

transmembrane domain

TM

transmembrane

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00619.

  • Additional 31P solid-state NMR spectra, simulations of spectral line shapes in the presence of motions and resulting topological analysis, and a more detailed analyses of the 2H and 15N spectral line shapes of Ala713 (PDF)

Author Contributions

§ A.I. and E.S.S. have contributed equally.

The authors declare no competing financial interest.

Footnotes

a

Due to different numbering schemes these correspond to G625, G629, G333, G634, and A638 in the solid-state NMR work of ref (23).

Supplementary Material

ao7b00619_si_001.pdf (2.1MB, pdf)

References

  1. Korsak M.; Kozyreva T. Beta Amyloid Hallmarks: From Intrinsically Disordered Proteins to Alzheimer’s Disease. Adv. Exp. Med. Biol. 2015, 870, 401–421. 10.1007/978-3-319-20164-1_14. [DOI] [PubMed] [Google Scholar]
  2. Musiek E. S.; Holtzman D. M. Three dimensions of the amyloid hypothesis: time, space and ‘wingmen’. Nat. Neurosci. 2015, 18, 800–806. 10.1038/nn.4018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Herrup K. The case for rejecting the amyloid cascade hypothesis. Nat. Neurosci. 2015, 18, 794–799. 10.1038/nn.4017. [DOI] [PubMed] [Google Scholar]
  4. Morris G. P.; Clark I. A.; Vissel B. Inconsistencies and controversies surrounding the amyloid hypothesis of Alzheimer’s disease. Acta Neuropathol. Commun. 2014, 2, 135. 10.1186/s40478-014-0135-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Qiu T.; Liu Q.; Chen Y. X.; Zhao Y. F.; Li Y. M. Abeta42 and Abeta40: similarities and differences. J. Pept. Sci. 2015, 21, 522–529. 10.1002/psc.2789. [DOI] [PubMed] [Google Scholar]
  6. Nguyen K. V. The human beta-amyloid precursor protein: biomolecular and epigenetic aspects. Biomol. Concepts 2015, 6, 11–32. 10.1515/bmc-2014-0041. [DOI] [PubMed] [Google Scholar]
  7. Agostinho P.; Pliassova A.; Oliveira C. R.; Cunha R. A. Localization and Trafficking of Amyloid-beta Protein Precursor and Secretases: Impact on Alzheimer’s Disease. J. Alzheimer’s Dis. 2015, 45, 329–347. 10.3233/JAD-142730. [DOI] [PubMed] [Google Scholar]
  8. Nhan H. S.; Chiang K.; Koo E. H. The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes. Acta Neuropathol. 2015, 129, 1–19. 10.1007/s00401-014-1347-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Nicolas M.; Hassan B. A. Amyloid precursor protein and neural development. Development 2014, 141, 2543–2548. 10.1242/dev.108712. [DOI] [PubMed] [Google Scholar]
  10. van der Kant R.; Goldstein L. S. Cellular functions of the amyloid precursor protein from development to dementia. Dev. Cell 2015, 32, 502–515. 10.1016/j.devcel.2015.01.022. [DOI] [PubMed] [Google Scholar]
  11. Nagase H.; Nakayama K. The Intracellular Domain of Amyloid Precursor Protein is a Potential Therapeutic Target in Alzheimer’s Disease. Curr. Drug Discovery Technol. 2014, 11, 243–258. 10.2174/1570163811666141121101358. [DOI] [PubMed] [Google Scholar]
  12. Kotler S. A.; Walsh P.; Brender J. R.; Ramamoorthy A. Differences between amyloid-beta aggregation in solution and on the membrane: insights into elucidation of the mechanistic details of Alzheimer’s disease. Chem. Soc. Rev. 2014, 43, 6692–6700. 10.1039/C3CS60431D. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Korshavn K. J.; Satriano C.; Lin Y.; Zhang R.; Dulchavsky M.; Bhunia A.; Ivanova M. I.; Lee Y. H.; La Rosa C.; Lim M. H.; Ramamoorthy A. Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-beta. J. Biol. Chem. 2017, 292, 4638–4650. 10.1074/jbc.M116.764092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Vassar R.; Bennett B. D.; Babu-Khan S.; Kahn S.; Mendiaz E. A.; Denis P.; Teplow D. B.; Ross S.; Amarante P.; Loeloff R.; Luo Y.; Fisher S.; Fuller J.; Edenson S.; Lile J.; Jarosinski M. A.; Biere A. L.; Curran E.; Burgess T.; Louis J. C.; Collins F.; Treanor J.; Rogers G.; Citron M. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 1999, 286, 735–741. 10.1126/science.286.5440.735. [DOI] [PubMed] [Google Scholar]
  15. Qi-Takahara Y.; Morishima-Kawashima M.; Tanimura Y.; Dolios G.; Hirotani N.; Horikoshi Y.; Kametani F.; Maeda M.; Saido T. C.; Wang R.; Ihara Y. Longer forms of amyloid beta protein: implications for the mechanism of intramembrane cleavage by gamma-secretase. J. Neurosci. 2005, 25, 436–445. 10.1523/JNEUROSCI.1575-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zhao G.; Mao G.; Tan J.; Dong Y.; Cui M.-Z.; Kim S.-H.; Xu X. Identification of a new presenilin-dependent zeta-cleavage site within the transmembrane domain of amyloid precursor protein. J. Biol. Chem. 2004, 279, 50647–50650. 10.1074/jbc.C400473200. [DOI] [PubMed] [Google Scholar]
  17. Krishnaswamy S.; Verdile G.; Groth D.; Kanyenda L.; Martins R. N. The structure and function of Alzheimer's gamma secretase enzyme complex. Crit. Rev. Clin. Lab. Sci. 2009, 46, 282–301. 10.3109/10408360903335821. [DOI] [PubMed] [Google Scholar]
  18. Scheuermann S.; Hambsch B.; Hesse L.; Stumm J.; Schmidt C.; Beher D.; Bayer T. A.; Beyreuther K.; Multhaup G. Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer’s disease. J. Biol. Chem. 2001, 276, 33923–33929. 10.1074/jbc.M105410200. [DOI] [PubMed] [Google Scholar]
  19. Miyashita N.; Straub J. E.; Thirumalai D.; Sugita Y. Transmembrane structures of amyloid precursor protein dimer predicted by replica-exchange molecular dynamics simulations. J. Am. Chem. Soc. 2009, 131, 3438–3439. 10.1021/ja809227c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Richter L.; Munter L.-M.; Ness J.; Hildebrand P. W.; Dasari M.; Unterreitmeier S.; Bulic B.; Beyermann M.; Gust R.; Reif B.; Weggen S.; Langosch D.; Multhaup G. Amyloid beta 42 peptide (Abeta42)-lowering compounds directly bind to Abeta and interfere with amyloid precursor protein (APP) transmembrane dimerization. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 14597–14602. 10.1073/pnas.1003026107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Barrett P. J.; Song Y.; Van Horn W. D.; Hustedt E. J.; Schafer J. M.; Hadziselimovic A.; Beel A. J.; Sanders C. R. The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol. Science 2012, 336, 1168–1171. 10.1126/science.1219988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nadezhdin K. D.; Bocharova O. V.; Bocharov E. V.; Arseniev A. S. Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment. FEBS Lett. 2012, 586, 1687–1692. 10.1016/j.febslet.2012.04.062. [DOI] [PubMed] [Google Scholar]
  23. Dominguez L.; Meredith S. C.; Straub J. E.; Thirumalai D. Transmembrane fragment structures of amyloid precursor protein depend on membrane surface curvature. J. Am. Chem. Soc. 2014, 136, 854–857. 10.1021/ja410958j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lu J.-X.; Yau W.-M.; Tycko R. Evidence from solid-state NMR for nonhelical conformations in the transmembrane domain of the amyloid precursor protein. Biophys. J. 2011, 100, 711–719. 10.1016/j.bpj.2010.12.3696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sato T.; Tang T. C.; Reubins G.; Fei J. Z.; Fujimoto T.; Kienlen-Campard P.; Constantinescu S. N.; Octave J. N.; Aimoto S.; Smith S. O. A helix-to-coil transition at the epsilon-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1421–1426. 10.1073/pnas.0812261106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Dominguez L.; Foster L.; Straub J. E.; Thirumalai D. Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, E5281–7. 10.1073/pnas.1606482113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Russ W. P.; Engelman D. M. The GxxxG motif: A framework for transmembrane helix-helix association. J. Mol. Biol. 2000, 296, 911–919. 10.1006/jmbi.1999.3489. [DOI] [PubMed] [Google Scholar]
  28. Munter L.-M.; Voigt P.; Harmeier A.; Kaden D.; Gottschalk K. E.; Weise C.; Pipkorn R.; Schaefer M.; Langosch D.; Multhaup G. GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42. EMBO J. 2007, 26, 1702–1712. 10.1038/sj.emboj.7601616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kienlen-Campard P.; Tasiaux B.; Van Hees J.; Li M.; Huysseune S.; Sato T.; Fei J. Z.; Aimoto S.; Courtoy P. J.; Smith S. O.; Constantinescu S. N.; Octave J.-N. Amyloidogenic processing but not amyloid precursor protein (APP) intracellular C-terminal domain production requires a precisely oriented APP dimer assembled by transmembrane GXXXG motifs. J. Biol. Chem. 2008, 283, 7733–7744. 10.1074/jbc.M707142200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sagi S. A.; Lessard C. B.; Winden K. D.; Maruyama H.; Koo J. C.; Weggen S.; Kukar T. L.; Golde T. E.; Koo E. H. Substrate sequence influences gamma-secretase modulator activity, role of the transmembrane domain of the amyloid precursor protein. J. Biol. Chem. 2011, 286, 39794–39803. 10.1074/jbc.M111.277228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mori T.; Miyashita N.; Im W.; Feig M.; Sugita Y. Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms. Biochim. Biophys. Acta 2016, 1858, 1635–1651. 10.1016/j.bbamem.2015.12.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Antzutkin O. N.; Balbach J. J.; Tycko R. Site-specific identification of non-beta-strand conformations in Alzheimer’s beta-amyloid fibrils by solid-state NMR. Biophys. J. 2003, 84, 3326–3335. 10.1016/S0006-3495(03)70057-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Goormaghtigh E.; Ruysschaert J. M.; Brasseur R.. Polarized Attenuated Total Reflection Infrared Spectroscopy as a Tool to Investigate the Conformation and Orientation of Membrane Components. In Molecular Description of Biological Membranes by Compouter Aided Conformational Analysis; Brasseur R., Ed.; CRC Press: Boca Raton, 1990; pp 285–329. [Google Scholar]
  34. Bechinger B.; Ruysschaert J. M.; Goormaghtigh E. Membrane Helix Orientation from Linear Dichroism of Infrared Attenuated Total Reflection Spectra. Biophys. J. 1999, 76, 552–563. 10.1016/S0006-3495(99)77223-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Bechinger B.; Sizun C. Alignment and structural analysis of membrane polypeptides by 15N and 31P solid-state NMR spectroscopy. Concepts Magn. Reson. 2003, 18A, 130–145. 10.1002/cmr.a.10070. [DOI] [Google Scholar]
  36. Bechinger B.; Resende J. M.; Aisenbrey C. The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: Established concepts and novel developments. Biophys. Chem. 2011, 153, 115–125. 10.1016/j.bpc.2010.11.002. [DOI] [PubMed] [Google Scholar]
  37. Aisenbrey C.; Bechinger B. Tilt and rotational pitch angles of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy. Biochemistry 2004, 43, 10502–10512. 10.1021/bi049409h. [DOI] [PubMed] [Google Scholar]
  38. Verly R. M.; de Moraes C. M.; Resende J. M.; Aisenbrey C.; Bemquemer M. P.; Pilo-Veloso D.; Valente A. P.; Alemida F. C.; Bechinger B. Structure and membrane interactions of the antibiotic peptide dermadistinctin k by solution and oriented 15 N and 31 P solid-state NMR spectroscopy. Biophys. J. 2009, 96, 2194–2203. 10.1016/j.bpj.2008.11.063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kim C.; Spano J.; Park E. K.; Wi S. Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3. Biochim. Biophys. Acta 2009, 1788, 1482–1496. 10.1016/j.bbamem.2009.04.017. [DOI] [PubMed] [Google Scholar]
  40. Holt A.; Rougier L.; Reat V.; Jolibois F.; Saurel O.; Czaplicki J.; Killian J. A.; Milon A. Order Parameters of a Transmembrane Helix in a Fluid Bilayer: Case Study of a WALP Peptide. Biophys. J. 2010, 98, 1864–1872. 10.1016/j.bpj.2010.01.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Michalek M.; Salnikov E. S.; Bechinger B. Structure and topology of the huntingtin 1–17 membrane anchor by a combined solution and solid-state NMR approach. Biophys. J. 2013, 105, 699–710. 10.1016/j.bpj.2013.06.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Osenkowski P.; Ye W.; Wang R.; Wolfe M. S.; Selkoe D. J. Direct and potent regulation of gamma-secretase by its lipid microenvironment. J. Biol. Chem. 2008, 283, 22529–22540. 10.1074/jbc.M801925200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Hartmann T.; Kuchenbecker J.; Grimm M. O. W. Alzheimer’s disease: the lipid connection. J. Neurochem. 2007, 103, 159–170. 10.1111/j.1471-4159.2007.04715.x. [DOI] [PubMed] [Google Scholar]
  44. Traaseth N. J.; Shi L.; Verardi R.; Muller D.; Barany G.; Veglia G. Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 10165–10170. 10.1073/pnas.0904290106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Resende J. M.; Moraes C. M.; Munhoz V. H. D. O.; Aisenbrey C.; Verly R. M.; Bertani P.; Cesar A.; Pilo-Veloso D.; Bechinger B. Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16639–16644. 10.1073/pnas.0905069106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Gorman P. M.; Kim S.; Guo M.; Melnyk R. A.; McLaurin J.; Fraser P. E.; Bowie J. U.; Chakrabartty A. Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer’s disease mutants. BMC Neurosci. 2008, 9, 17. 10.1186/1471-2202-9-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Eggert S.; Midthune B.; Cottrell B.; Koo E. H. Induced dimerization of the amyloid precursor protein leads to decreased amyloid-beta protein production. J. Biol. Chem. 2009, 284, 28943–28952. 10.1074/jbc.M109.038646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ramamoorthy A.; Wei Y.; Lee D. PISEMA Solid-State NMR Spectroscopy. Annu. Rep. NMR Spectrosc. 2004, 52, 1–52. 10.1016/S0066-4103(04)52001-X. [DOI] [Google Scholar]
  49. Bai X.-c.; Yan C.; Yang G.; Lu P.; Ma D.; Sun L.; Zhou R.; Scheres S. H. W.; Shi Y. An atomic structure of human gamma-secretase. Nature 2015, 525, 212–217. 10.1038/nature14892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Chan W. C.; White S. H.. Fmoc Solid-Phase Peptide Synthesis: A Practical Approach; Oxford University Press: Oxford, 2000. [Google Scholar]
  51. Andrushchenko V. V.; Vogel H. J.; Prenner E. J. Optimization of the hydrochloric acid concentration used for trifluoroacetate removal from synthetic peptides. J. Pept. Sci. 2007, 13, 37–43. 10.1002/psc.793. [DOI] [PubMed] [Google Scholar]
  52. Goormaghtigh E.; Cabiaux V.; Ruysschaert J. M. Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds. Sub-Cell. Biochem. 1994, 23, 329–362. 10.1007/978-1-4615-1863-1_8. [DOI] [PubMed] [Google Scholar]
  53. Fringeli U. P.; Gunthard H. H. Infrared membrane spectroscopy. Mol. Biol. Biochem. Biophys. 1981, 31, 270–332. 10.1007/978-3-642-81537-9_6. [DOI] [PubMed] [Google Scholar]
  54. Aisenbrey C.; Bertani P.; Bechinger B.. Solid-State NMR Investigations of Membrane-Associated Antimicrobial Peptides. In Antimicrobial Peptides; Guiliani A., Rinaldi A. C., Eds.; Humana Press: N.Y., 2010; pp 209–233. [DOI] [PubMed] [Google Scholar]
  55. Rance M.; Byrd R. A. Obtaining High-Fidelity Spin-1/2 Powder Spectra in Anisotropic Media: Phase-Cycled Hahn Echo Spectroscopy. J. Magn. Reson. 1983, 52, 221–240. 10.1016/0022-2364(83)90190-7. [DOI] [Google Scholar]
  56. Bechinger B.; Opella S. J. Flat-Coil Probe for NMR Spectroscopy of Oriented Membrane Samples. J. Magn. Reson. 1991, 95, 585–588. 10.1016/0022-2364(91)90173-Q. [DOI] [Google Scholar]
  57. Hediger S.; Meier B. H.; Ernst R. R. Adiabatic passage Hartmann-Hahn cross polarization in NMR under magic angle sample spinning. Chem. Phys. Lett. 1995, 240, 449–456. 10.1016/0009-2614(95)00505-X. [DOI] [Google Scholar]
  58. Bertani P.; Raya J.; Bechinger B. 15N chemical shift referencing in solid state NMR. Solid State Nucl. Magn. Reson. 2014, 61–62, 15–18. 10.1016/j.ssnmr.2014.03.003. [DOI] [PubMed] [Google Scholar]
  59. Davis J. H.; Jeffrey K. R.; Bloom M.; Valic M. I.; Higgs T. P. Quadrupolar Echo Deuteron Magnetic Resonance Spectroscopy in Ordered Hydrocarbon Chains. Chem. Phys. Lett. 1976, 42, 390–394. 10.1016/0009-2614(76)80392-2. [DOI] [Google Scholar]
  60. Salnikov E.; Bertani P.; Raap J.; Bechinger B. Analysis of the amide (15)N chemical shift tensor of the C(alpha) tetrasubstituted constituent of membrane-active peptaibols, the alpha-aminoisobutyric acid residue, compared to those of di- and tri-substituted proteinogenic amino acid residues. J. Biomol. NMR 2009, 45, 373–387. 10.1007/s10858-009-9380-5. [DOI] [PubMed] [Google Scholar]
  61. Batchelder L. S.; Niu H.; Torchia D. A. Methyl reorientation in polycrystalline amino acids and peptides: A 2H NMR spin lattice relaxation study. J. Am. Chem. Soc. 1983, 105, 2228–2231. 10.1021/ja00346a021. [DOI] [Google Scholar]
  62. Mendonca de Moraes C.; Bechinger B. Peptide-related alterations of membrane-associated water: Deuterium solid-state NMR investigations of phosphatidylcholine membranes at different hydration levels. Magn. Reson. Chem. 2004, 42, 155–161. 10.1002/mrc.1321. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao7b00619_si_001.pdf (2.1MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES