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Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18–Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, respressor or co-activator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behaviour of the cytoplasmic DNA binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly-defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a 'DNA-catcher' to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.

Significance We uncovered a molecular mechanism by which multiple importins regulate nuclear import, phase separation, and stress granule recruitment of an RNA-binding protein. Our work permitted the identification of two types of nuclear localization signal (NLS) in cold-inducible RNA-binding protein (CIRBP). We show that CIRBP, which lacks any classical NLS, binds both the importin Transportin-1 (TNPO1) and Transportin-3 (TNPO3). These interactions and nuclear import of CIRBP involve two distinct CIRBP regions, the arginine-glycine(-glycine) (RG/RGG) region and the discovered arginine-serine-tyrosine (RSY)–rich region. We show that these two regions harbor different specificity toward TNPO1 and TNPO3, with the RG/RGG-NLS being specialized in TNPO1 recognition and the RSY-NLS being specialized in TNPO3 recognition., The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils., Graphical abstract Image 1, Highlights • The only cysteine in human p16INK4A is prone to oxidation in vitro and in vivo. • p16INK4A forms disulfide-dependent homodimers upon oxidation. • The all α-helical p16INK4A protein aggregates as β-amyloids upon oxidation. • Oxidation and subsequent β-amyloid formation impair p16INK4A function.

Nuclear magnetic resonance (NMR) spectroscopy is a well suited method for the analysis of biomolecules in solution and provides unique insights into their structure, dynamics and function. Recent work has provided new approaches to determine surface-accessibility of biomolecules through the addition of freely soluble paramagnetic agents. This pushes the current limits towards more accurate and reliable structures, faster data acquisition and the detailed analysis of dynamic processes.

Die Erforschung von intrinsisch unstrukturierten Proteinen (IDPs) mit NMR-spektroskopischen Methoden wird oftmals durch starke Uberlagerung der Proteinsignale erschwert. Dies fuhrt zu uneindeutigen Zuordnungen dieser Signale und erschwert die Datenanalyse, in welcher oftmals eine gute Separierung der Signale die Grundlage der Dateninterpretation bildet. In dieser Arbeit berichten wir von der Moglichkeit, einen Lanthanoidkomplex kovalent an das Zielprotein zu binden, der zu einer erhohten Dispersion der Signale fuhrt. Wir zeigen diesen Effekt an Proteinen mit sich wiederholenden Aminosauresequenzen, welche somit besonders von Signaluberlappung betroffen sind. Die Bindung eines DOTA-basierten Lanthanoidkomplexes an ein Cystein fuhrt zu pseudochemischen Verschiebungen (PCS), die noch in mehr als 20 Aminosaureresten entfernten Regionen detektierbar sind. Die stark erhohte Dispersion der beobachteten Signale fuhrt zu einer betrachtlichen Erleichterung in der Zuordnung und Datenauswertung.

An approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance‐to‐surface information encoded in the sPRE data in the chemical shift‐based CS‐Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach.

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.

A novel role for S100B, a recognized brain distress marker, as a chaperone-like suppressor of Aβ42 aggregation and toxicity., Amyloid-β (Aβ) aggregation and neuroinflammation are consistent features in Alzheimer’s disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aβ42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aβ42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aβ42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aβ42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aβ42 aggregation by interacting with Aβ42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface–catalyzed reactions through S100B binding to growing Aβ42 oligomers and fibrils. S100B protects cells from Aβ42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aβ42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.

The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (V-L) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the VL and CL domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of VL and CL to each other are of major importance for maintaining the structural integrity of the full-length LC. (C) 2018 Elsevier Ltd. All rights reserved.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

NMR spectroscopy is a key method for studying the structure and dynamics of (large) multidomain proteins and complexes in solution. It plays a unique role in integrated structural biology approaches as especially information about conformational dynamics can be readily obtained at residue resolution. Here, we review NMR techniques for such studies focusing on state-of-the-art tools and practical aspects. An efficient approach for determining the quaternary structure of multidomain complexes starts from the structures of individual domains or subunits. The arrangement of the domains/subunits within the complex is then defined based on NMR measurements that provide information about the domain interfaces combined with (long-range) distance and orientational restraints. Aspects discussed include sample preparation, specific isotope labeling and spin labeling; determination of binding interfaces and domain/subunit arrangements from chemical shift perturbations (CSP), nuclear Overhauser effects (NOEs), isotope editing/filtering, cross-saturation, and differential line broadening; and based on paramagnetic relaxation enhancements (PRE) using covalent and soluble spin labels. Finally, the utility of complementary methods such as small-angle X-ray or neutron scattering (SAXS, SANS), electron paramagnetic resonance (EPR) or fluorescence spectroscopy techniques is discussed. The applications of NMR techniques are illustrated with studies of challenging (high molecular weight) protein complexes.

This is the first study determining the three-dimensional structure of Phl p 5a which reveals a novel mechanism for high allergenic activity based on flexibly connected IgE-reactive domains which cross-link effector cell-bound IgE more efficiently than isolated rigid globular proteins. These findings may also form a basis for specific immunotherapy strategies.

The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical‐shift assignments, and data analysis that relies on well‐separated resonances. We present a covalent paramagnetic lanthanide‐binding tag (LBT) for increasing the chemical‐shift dispersion and facilitating the chemical‐shift assignment of challenging, repeat‐containing IDPs. Linkage of the DOTA‐based LBT to a cysteine residue induces pseudo‐contact shifts (PCS) for resonances more than 20 residues from the spin‐labeling site. This leads to increased chemical‐shift dispersion and decreased signal overlap, thereby greatly facilitating chemical‐shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat‐containing IDPs and low‐complexity regions. This results in improved efficiency for IDP analysis and binding studies.

Lanthanide complexes based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) cage are commonly used as phase contrast agents in magnetic resonance imaging, but can also be utilized in structural NMR applications due to their ability to induce either paramagnetic relaxation enhancement or a pseudo-contact shift (PCS) depending on the choice of the lanthanide. The size and sign of the PCS for any given atom is determined by its coordinates relative to the metal center, and the characteristics of the lanthanide’s magnetic susceptibility tensor. Using a polymethylated DOTA tag (Ln-M8-SPy) conjugated to ubiquitin, we calculated the position of the metal center and characterized the susceptibility tensor for a number of lanthanides (dysprosium, thulium, and ytterbium) under a range of pH and temperature conditions. We found that there was a difference in temperature sensitivity for each of the complexes studied, which depended on the size of the lanthanide ion as well as the isomeric state of the cage. Using (17)O-NMR, we confirmed that the temperature sensitivity of the compounds was enhanced by the presence of an apically bound water molecule. Since amide-containing lanthanide complexes are known to be pH sensitive and can be used as probes of physiological pH, we also investigated the effect of pH on the Ln-M8-SPy susceptibility tensor, but we found that the changes in this pH range (5.0–7.4) were not significant.

The par toxin-antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin-antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin-antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding alpha-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, alpha-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.

The interaction with biological membranes is of functional importance for many peptides and proteins. Structural studies on such membrane-bound biomacromolecules are often carried out in solutions containing small membrane-mimetic assemblies of detergent molecules. To investigate the influence of the hydrophobic chain length on the structure, diffusional and dynamical behavior of a peptide bound to micelles, we studied the binding of three peptides to n-phosphocholines with n ranging from 8 to 16. The peptides studied are the 15 residue antimicrobial peptide CM15, the 25-residue transmembrane helix 7 of yeast V-ATPase (TM7), and the 35-residue bacterial toxin LdrD. To keep the dimension of the peptide-membrane-mimetic assembly small, micelles are typically used when studying membrane-bound peptides and proteins, for example, by solution NMR spectroscopy. Since they are readily available in deuterated form most often sodium-dodecylsulfate (SDS) and dodecylphosphocholine (DPC) are used as the micelle-forming detergent. Using NMR, CD, and SAXS, we found that all phosphocholines studied form spherical micelles in the presence and absence of small bound peptides and the diameters of the micelles are basically unchanged upon peptide binding. The size of the peptide relative to the micelle determines to what extent the secondary structure can form. For small peptides (up to approximately 25 residues) the use of shorter chain phosphocholines is recommended for solution NMR studies due to the favorable spectral quality and since they are as well-structured as in DPC. In contrast, larger peptides are better structured in micelles formed by detergents with chain lengths longer than DPC.

Deposition of the nuclear DNA/RNA-binding protein Fused in sarcoma (FUS) in cytosolic inclusions is a common hallmark of some cases of frontotemporal lobar degeneration (FTLD-FUS) and amyotrophic lateral sclero- sis (ALS-FUS). Whether both diseases also share common pathological mechanisms is currently unclear. Based on our previous finding that FUS deposits are hypomethylated in FTLD-FUS but not in ALS-FUS, we have now investigated whether genetic or pharmacological inactivation of Pro- tein arginine methyltransferase 1 (PRMT1) activity results in unmethylated FUS or in alternatively methylated forms of FUS. To do so, we generated FUS-specific monoclonal antibodies that specifically recognize unmethylated argi- nine (UMA), monomethylated arginine (MMA) or asym- metrically dimethylated arginine (ADMA). Loss of PRMT1 indeed not only results in an increase of UMA FUS and a decrease of ADMA FUS, but also in a significant increase of MMA FUS. Compared to ADMA FUS, UMA and MMA FUS exhibit much higher binding affinities to Transportin-1, the nuclear import receptor of FUS, as measured by pull- down assays and isothermal titration calorimetry. Moreover, we show that MMA FUS occurs exclusively in FTLD-FUS, but not in ALS-FUS. Our findings therefore provide addi- tional evidence that FTLD-FUS and ALS-FUS are caused by distinct disease mechanisms although both share FUS deposits as a common denominator.

Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

Interactions of macrolide antibiotics with biological membranes contribute to their bioavailability but are also involved in the formation of phospholipidosis, which is caused by the inhibition of phospholipase A(1) activity. We determined the interaction strength and localization of macrolide antibiotics with membrane-mimetics. Macrolides bind to membrane-mimetics with the positively charged amino groups being close to the micelle surface and thereby protect the lipids from being degraded by phospholipase A(1) rather than inhibiting the enzyme.

Many peptides, proteins, and drugs interact with biological membranes, and knowing the mode of binding is essential to understanding their biological functions. To obtain the complete orientation and immersion depth of such a compound, the membrane-mimetic system (micelle) is placed in an aqueous buffer containing the soluble and inert paramagnetic contrast agent Gd(DTPA-BMA). Paramagnetic relaxation enhancements (PREs) of a specific nucleus then depend only on its distance from the surface. The positioning of a structurally characterized compound can be obtained by least-squares fitting of experimental PREs to the micelle center position. This liquid-state NMR approach, which does not rely on isotopic labeling or chemical modification, has been applied to determine the location of the presumed transmembrane region 7 of yeast V-ATPase (TM7) and the membrane-bound antimicrobial peptide CM15 in micelles. TM7 binds in a trans-micelle orientation with the N-terminus being slightly closer to the surface than the C-terminus. CM15 is immersed unexpectedly deep into the micelle with the more hydrophilic side of the helix being closer to the surface than the hydrophobic one.

Many antimicrobial peptides form alpha-helices when bound to a membrane. In addition, around 80% of residues in membrane-bound proteins are found in alpha-helical regions. The orientation and location of such helical peptides and proteins in the membrane are key factors determining their function and activity. Here we present a new solution state NMR method for obtaining the orientation of helical peptides in a membrane-mimetic environment (micelle-bound) without any chemical perturbation of the peptide-micelle system. By monitoring proton longitudinal relaxation rates upon addition of the freely water-soluble and inert paramagnetic probe Gd(DTPA-BMA) to an alpha-helical peptide, a wavelike pattern with a periodicity of 3.6 residues per turn is observed. The tilt and azimuth (rotation) angle of the helix determine the shape of this paramagnetic relaxation wave and can be obtained by least-square fitting of measured relaxation enhancements. Results are presented for the 15-residue antimicrobial peptide CM15 which forms an amphipathic helix almost parallel to the surface of the micelle. Thus, a few fast experiments enable the identification of helical regions and determination of the helix orientation within the micelle without the need for covalent modification, isotopic labeling, or sophisticated equipment. This approach opens a path toward the topology determination of alpha-helical membrane-proteins without the need for a complete NOE-based structure determination.