Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 3₁₀-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 3₁₀-helical region of the substrate’s TMD. This 3₁₀-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 3₁₀-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.     

Truncated hemoglobins: trimming the classical ‘three-over-three’ globin fold to a minimal size

Truncated hemoglobins (trHbs) host the heme in a “two-over-two’ α-helical sandwich which results from extensive editing of the classical ‘three-over-three’ globin fold. The three-dimensional structure of trHbs is based on four main α-helices, arranged in a sort of α-helical bundle composed of two antiparallel helix pairs (B/E and G/H). Most notably, trHbs deviate from the conventional globin fold in that they display an extended loop substituting for the heme proximal F-helix observed in globins. Moreover, since efficient adaptation of a 110–130 amino acid trHb chain to host the porphyrin ring firstly requires specific chain flexibility, trHbs contain three invariant Gly-based motifs. Inspection of the trHb three-dimensional trHb structures shows that an apparent protein cavity or tunnel would connect the protein surface to an inner region very close to the heme distal site. Such a structural feature, never observed before in (non) vertebrate globins, may have substantial implications for ligand diffusion and binding properties in trHbs.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α_1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

Contribution of Long-Range Interactions to the Secondary Structure of an Unfolded Globin

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an α-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable α-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins.     

Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel

Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane α-helical bundles, related by a ∼50° rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM α-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric α-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function.     

Energetics and structure of alanine-rich α-helices via adaptive steered molecular dynamics

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich α-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA)_kF-NH₂ and a natural α-helical peptide a2N (1–17) provide evidence of the hypothesis’s generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alanine-rich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the α-helical peptides results in a turnover mechanism in which a 3₁₀-helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich α-helical peptides in between the endpoints—namely the native structure and free coil—is nearly independent of the length or the specific primary structure.     

How Does a Simplified-Sequence Protein Fold?

To investigate a putatively primordial protein we have simplified the sequence of a 56-residue α/β fold (the immunoglobulin-binding domain of protein G) by replacing it with polyalanine, polythreonine, and diglycine segments at regions of the sequence that in the folded structure are α-helical, β-strand, and turns, respectively. Remarkably, multiple folding and unfolding events are observed in a 15-μs molecular dynamics simulation at 330 K. The most stable state (populated at ∼20%) of the simplified-sequence variant of protein G has the same α/β topology as the wild-type but shows the characteristics of a molten globule, i.e., loose contacts among side chains and lack of a specific hydrophobic core. The unfolded state is heterogeneous and includes a variety of α/β topologies but also fully α-helical and fully β-sheet structures. Transitions within the denatured state are very fast, and the molten-globule state is reached in <1 μs by a framework mechanism of folding with multiple pathways. The native structure of the wild-type is more rigid than the molten-globule conformation of the simplified-sequence variant. The difference in structural stability and the very fast folding of the simplified protein suggest that evolution has enriched the primordial alphabet of amino acids mainly to optimize protein function by stabilization of a unique structure with specific tertiary interactions.     

The Relation between α-Helical Conformation and Amyloidogenicity

Amyloid fibrils are stable aggregates of misfolded proteins and polypeptides that are insoluble and resistant to protease activity. Abnormal formation of amyloid fibrils in vivo may lead to neurodegenerative disorders and other systemic amyloidosis, such as Alzheimer’s, Parkinson’s, and atherosclerosis. Because of their clinical importance, amyloids are under intense scientific research. It is believed that short polypeptide segments within proteins are responsible for the transformation of correctly folded proteins into parts of larger amyloid fibrils and that this transition is modulated by environmental factors, such as pH, salt concentration, interaction with the cell membrane, and interaction with metal ions. Most studies on amyloids focus on the amyloidogenic sequences. The focus of this study is on the structure of the amyloidogenic α-helical segments because the α-helical secondary structure has been recognized to be a key player in different stages of the amyloidogenesis process. We have previously shown that the α-helical conformation may be expressed by two parameters (θ and ρ) that form orthogonal coordinates based on the Ramachandran dihedrals (φ and ψ) and provide an illuminating interpretation of the α-helical conformation. By performing statistical analysis on α-helical conformations found in the Protein Data Bank, an apparent relation between α-helical conformation, as expressed by θ and ρ, and amyloidogenicity is revealed. Remarkably, random amino acid sequences, whose helical structures were obtained from the most probable dihedral angles, revealed the same dependency of amyloidogenicity, suggesting the importance of α-helical structure as opposed to sequence.     

Sequential Unfolding of Beta Helical Protein by Single-Molecule Atomic Force Microscopy

The parallel βhelix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large ‘TpsA’ family all fold into long βhelices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the βhelix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the βhelix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.     

Helix-helix interactions and their impact on protein motifs and assemblies

Protein secondary structure elements are arranged in distinct structural motifs such as four-α-helix bundle, 8α/8β TIM-barrel, Rossmann dinucleotide binding fold, assembly of a helical rod. Each structural motif is characterized by a particular type of helix-helix interactions. A unique pattern of contacts is formed by interacting helices of the structural motif. In each type of fold, edges of the helix surface, which participate in the formation of helix-helix contacts with preceding and following helices, differ. This work shows that circular arrangements of the four, eight, and sixteen α-helices, which are found in the four-α-helical motif, TIM-barrel 8α/8β fold, and helical rod of 16.3¯ helices per turn correspondingly, can be associated with the mutual positioning of the edges of the helix surfaces. Edges (i, i+1)−(i+1, i+2) of the helix surface are central for the interhelical contacts in a four-α-helix bundle. Edges (i, i+1)−(i+2, i+3) are involved in the assembly of four-α-helix subunits into helical rod of a tobacco mosaic virus and a three-helix fragment of a Rossmann fold. In 8α/8β TIM-barrel fold, edges (i, i+1)−(i+5, i+6) are involved in the octagon arrangement. Approximation of a cross section of each motif with a polygon (n-gon, n=4, 8, 16) shows that a good correlation exists between polygon interior angles and angles formed by the edges of helix surfaces.     

Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the α-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the α-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an “α-helix-extended segment-α-helix” structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the α-helical folding. Both histidines are buried in SDS micelles as evidenced by their pK_a values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an α-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific “α-helix-extended segment-α-helix” structure of TM6 may have an important implication for the binding of the transporter to H⁺ and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.     

Light Signal Transduction Pathway from Flavin Chromophore to the Jα Helix of Arabidopsis Phototropin1

In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jα helix) for the function of phototropin; however, it remains unclear how the Jα helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jα) of the Jα helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (−)/1625 (+) cm⁻¹) of LOV2-Jα at physiological temperatures (≥260 K), corresponding to structural perturbation of the α-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the β-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jα helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jα helix in Arabidopsis phot1.     

Synthesis and characterization of poly(LysAla3)

The synthesis and characterization of poly(LysAla₃) are described. The polytetrapeptide is a model for short sequences found in proelastin, and is presumably involved in desmosine or isodesmosine cross-link formation in the native protein. Poly(LysAla₃) is found to possess a mixture of conformations in aqueous solution dependent on molecular weight and pH. Low-molecular-weight (ca. 3000) material appears to be a mixture of random and extended helix at neutral pH. However, as the molecular weight is increased an increasing amount of α-helix is observed rising to >50% for mol wt = 21,000. The α-helical chain segments are thermally stable, melting to a mixture of extended and random forms at T_m = 25°C. High pH (10.5) promotes further α-helix formation but at pH >11.0 the polypeptide becomes insoluble. The inference is that short chain segments of the peptide in elastin are unlikely to be α-helical in the equilibrium state but may fluctuate through such a conformation.     

Effects of Hofmeister ions on the α-helical structure of proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO₄ in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO₄ originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO₄ denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.     

Membrane-induced folding and structure of membrane-bound annexin A1 N-terminal peptides: implications for annexin-induced membrane aggregation

Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an α-helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an α-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into α-helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor.     

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 3₁₀ conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 3₁₀ conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 3₁₀ conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-3₁₀ helical conversions of the S4 in Ci-VSP associated with voltage activation.     

Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states

The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for anα-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to beα-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energyα-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in theirα-helical and bend conformations shows that the all-α-helical dimer is clearly favored energetically over the bend dimer.     

Structure and Topology of the Huntingtin 1–17 Membrane Anchor by a Combined Solution and Solid-State NMR Approach

The very amino-terminal domain of the huntingtin protein is directly located upstream of the protein’s polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntington’s disease. This huntingtin 1–17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1–17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1–17 labeled with ¹⁵N and ²H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1–17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1–17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1–17 domains and possibly with the proximal polyglutamine tract.