Heteronuclear NMR Experiments Research Articles

Structure of prolylrapamycin: confirmation through a revised and detailed NMR assignment study.

A complete and detailed characterization of Rapamycin (1) and Prolylrapamycin (2) has been conducted by homo- and hetero-nuclear NMR experiments in DMSO-d6 along with HRMS and FT-IR spectra and DSCs analyses. The NMR experiments allowed the assignment of every single proton and carbon atom belonging to the two structures and the definitive confirm of the presence of a pyrrolidine ring in Prolylrapamycin (2) in place of the piperidine ring that characterizes the structure of Sirolimus.

In-Cell 19F NMR of Proteins: Recent Progress and Future Opportunities.

In vitro, 19F NMR methodology is preferably selected as a complementary and straightforward method for unveiling the conformations, dynamics, and interactions of biological molecules. Its effectiveness in vivo has seen continuous improvement, addressing challenges faced by conventional heteronuclear NMR experiments on structured proteins, such as severe line broadening, low signal-to-noise ratio, and background signals. Herein, we summarize the distinctive advantages of 19F NMR, along with recent progress in sample preparation and applications within the realm of in-cell NMR. Additionally, we offer insights into the future directions and prospects of this methodology based on our understanding.

Multinuclear 1H/13C/15N chemical shift assignment of therapeutic octreotide acetate performed at natural abundance.

Octreotide acetate, the active pharmaceutical ingredient in the long-acting release (LAR) drug product Sandostatin®, is a cyclic octapeptide that mimics the naturally occurring somatostatin peptide hormone. Modern NMR can be a robust analytical method to identify and quantify octreotide molecules. Previous 1H chemical shift assignments were mostly performed in organic solvents, and no assignments for heteronuclear 13C, 15N, and aromatic 1H nuclei are available. Here, using state-of-the-art 1D and 2D homo- and heteronuclear NMR experiments, octreotide was fully assigned, including water exchangeable amide protons, in aqueous buffer except for 13CO and 15NH of F1, 15NH of C2, and 15NζHζ of K5 that were not observed because of water exchange or conformational exchange. The solution NMR spectra were then directly compared with 1D 1H/13C/15N solid-state NMR (SSNMR) spectra showing the potential applicability of 13C/15N SSNMR for octreotide drug product characterization.

Conformational Preferences at the Glycosidic Linkage of Saccharides in Solution as Deduced from NMR Experiments and MD Simulations: Comparison to Crystal Structures.

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles φ and ψ have been investigated for O-methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in α-l-Fucp-(1→3)-β-d-Glcp-OMe, α-l-Fucp-(1→3)-α-d-Galp-OMe and α-d-Glcp-(1→4)-α-d-Galp-OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one-dimensional 1 H,1 H-NOESY NMR experiments to obtain transglycosidic proton-proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3 JCH transglycosidic coupling constants related to torsion angles φ and ψ. Computed effective proton-proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the α-(1→3)-linked disaccharides and revealed that for the bimodal distribution at the ψ torsion angle for the α-(1→4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter-residue hydrogen bonding pattern for the α-(1→4)-linked disaccharide, including a nonconventional hydrogen bond between H5' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1 H NMR chemical shift displacement compared to α-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles φ and ψ in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation.

AI-designed RF pulses enable fast pulsing heteronuclear multiple quantum coherence NMR experiment at high and ultra-high magnetic fields.

We present a new AI-optimized 2D heteronuclear multiple quantum coherence (RAPID-HMQC) pulse sequence for NMR spectroscopy. RAPID-HMQC is a longitudinal 1H relaxation-optimized experiment with new AI-designed band-selective pulses to accelerate the analysis of organic compounds, metabolites, biopolymers, and real-time monitoring of dynamic processes at high- and ultra-high magnetic fields.

Serendipitous N,S-difunctionalization of triazoles with trifluoromethyl-β-diketones: access to regioisomeric 1-trifluoroacetyl-3-aryl-5-(2-oxo-2-arylethylthio)-1,2,4-triazoles as DNA-groove binders.

In the present research work, a serendipitous regioselective synthesis of DNA targeting agents, 1-trifluoroacetyl-3-aryl-5-(2-oxo-2-arylethylthio)-1,2,4-triazoles, has been achieved through the one-pot cascade reaction of 3-mercapto[1,2,4]triazoles with trifluoromethyl-β-diktetones in presence of NBS instead of the cyclized thiazolo[3,2-b][1,2,4]triazole. The present protocol offered a unique approach for functionalizing both N-acylation and S-alkylation in a concerted fashion. The structures of the regioisomeric products were thoroughly characterized by heteronuclear 2D NMR experiments. Facile scalability and excellent atom economy through easily available starting reactants are the notable features of the present sustainable protocol. Targeting tumor cell DNA with minor groove-binding small molecules has proven highly effective in the recent past, drawing significant attention for combating tumor-related afflictions. In this context, the synthesized analogs were primarily screened for their ability to bind with the DNA duplex d(CGCGAATTCGCG)2 using molecular modeling tools. Additionally, the most promising compound 14m was deployed as a probe for DNA sensing and interaction mechanisms with calf thymus (ct)DNA through various spectral techniques at a physiologic temperature of 37 °C. It has been found that the compound demonstrated a strong binding affinity (Kb = 1 × 105 M-1) with double-helical DNA, particularly within the minor groove, resulting in the formation of a stable complex through static quenching (Kq = 5.86 ± 0.11 × 1012 M-1 s-1). The fluorescent displacement assay confirmed that the quencher binds to the minor groove of ctDNA, further supported by circular dichroism and viscosity studies.

Two new tirucallane-type triterpenes from the aerial parts of Euphorbia retusa

Chemical characterization of the dichloromethane extract of Euphorbia retusa aerial parts by chromatographic methods afforded an inseparable mixture of two new tirucallane-type triterpenoids namely (3β, 24S)- and (3β, 24R)-tirucalla-7,25-diene-3,24-diol (1a and 1b) respectively, together with nine known compounds including six triterpenoids and three phenylpropanoids. Their structures were established on the basis of spectroscopic analyses, including 1D- and 2D- homo- and heteronuclear NMR experiments and mass spectrometry, as well as comparison with data from related compounds. The chemotaxonomic aspects were also discussed.

Optimal control derived sensitivity-enhanced CA-CO mixing sequences for MAS solid-state NMR – Applications in sequential protein backbone assignments

We have recently introduced optimal-control derived pulse sequences for sensitivity-enhanced heteronuclear correlation NMR experiments of solid proteins. Preservation of equivalent coherence transfer pathways using transverse-mixing pulses (TROP) in multidimensional pulse schemes allows to increase the sensitivity of the experiments by more than a factor of 2 per each indirect dimension. In this article, we present homonuclear CA-CO transverse-mixing elements (homoTROP) that are based on dipolar interactions and achieve similar gains as the heteronuclear TROP pulses described previously. Both transfer elements were subsequently implemented in 3D se-hCAcoNH and se-hCOcaNH, that together with the previously introduced 3D se-hCANH and se-hCONH experiments yield a complete set of sensitivity-enhanced protein backbone assignment experiments. In contrast to the J-coupling based methods that are used at fast (60 kHz) and ultrafast MAS (>100 kHz), the homoTROP experiments employ about 10-times shorter mixing times making use of the larger magnitude of the dipolar coupling in comparison to the J couplings. The experiments are demonstrated using a microcrystalline, perdeuterated sample of the chicken alpha-spectrin SH3 domain in which all exchangeable sites are fully back-substituted with protons. We evaluated the gains in efficiency in all experiments site-specifically observing that the se-hCAcoNH and se-hCOcaNH experiments yield an increase in sensitivity by a factor of 1.36±0.09 and at least a factor of 1.8 with respect to the conventional hcoCAcoNH and hCOcaNH J-based experiments.

An expeditious on-water regioselective synthesis of novel arylidene-hydrazinyl-thiazoles as DNA targeting agents

A series of twenty novel (E)-arylidene-hydrazinyl-thiazole derivatives has been synthesized employing α-bromo-β-diketones, thiosemicarbazide, and aromatic/heteroaromatic aldehydes with a simple and facile one-pot multicomponent reaction passageway. This organic transformation proceeds efficiently in aqueous media and demonstrated a large functional group tolerance. The structures and stereochemistry of the regioisomeric product were rigorously characterized using heteronuclear 2D NMR experiments. The binding potential of the synthesized analogs with B-DNA dodecamer d(CGCGAATTCGCG)2 was primarily screened using molecular modeling tools and further, mechanistic investigations (either groove or intercalation) were performed using various spectroscopic techniques such as UV–Visible, Fluorescence, and Circular dichroism. The absorption spectra showed a hyperchromic shift in the absorption maxima of ctDNA with successive addition of thiazole derivatives, implying groove binding mode of interactions, further supported by displacement assay and circular dichroism analysis. Furthermore, steady-state fluorescence analysis revealed the static mode of quenching and moderate bindings between the ligand and DNA biomolecule. The competitive studies showed that the derivatives having a pyridinyl (heteroaromatic) group in their structure, bind with the nucleic acid of calf-thymus (ctDNA) more effectively in the minor groove region as compared with the aromatic substitutions.

Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy.

In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments. 19F NMR can overcome this issue, as fluorine atoms incorporated in proteins can be detected by simple background-free 1D NMR spectra. Here, we show that fluorinated amino acids can be easily incorporated in proteins expressed in human cells by employing a medium switch strategy. This straightforward approach allows the incorporation of different fluorinated amino acids in the protein of interest, reaching fluorination efficiencies up to 60%, as confirmed by mass spectrometry and X-ray crystallography. The versatility of the approach is shown by performing 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N in-cell NMR. We apply the approach to observe the interaction between an intracellular target, carbonic anhydrase 2, and its inhibitors, and to investigate how the formation of a complex between superoxide dismutase 1 and its chaperone CCS modulates the interaction of the chaperone subunit with the cellular environment.

Nuclear Magnetic Resonance and Metadynamics Simulations Reveal the Atomistic Binding of l-Serine and O-Phospho-l-Serine at Disordered Calcium Phosphate Surfaces of Biocements

Interactions between biomolecules and structurally disordered calcium phosphate (CaP) surfaces are crucial for the regulation of bone mineralization by noncollagenous proteins, the organization of complexes of casein and amorphous calcium phosphate (ACP) in milk, as well as for structure–function relationships of hybrid organic/inorganic interfaces in biomaterials. By a combination of advanced solid-state NMR experiments and metadynamics simulations, we examine the detailed binding of O-phospho-l-serine (Pser) and l-serine (Ser) with ACP in bone-adhesive CaP cements, whose capacity of gluing fractured bone together stems from the close integration of the organic molecules with ACP over a subnanometer scale. The proximity of each carboxy, aliphatic, and amino group of Pser/Ser to the Ca2+ and phosphate species of ACP observed from the metadynamics-derived models agreed well with results from heteronuclear solid-state NMR experiments that are sensitive to the 13C–31P and 15N–31P distances. The inorganic/organic contacts in Pser-doped cements are also contrasted with experimental and modeled data on the Pser binding at nanocrystalline HA particles grown from a Pser-bearing aqueous solution. The molecular adsorption is driven mainly by electrostatic interactions between the negatively charged carboxy/phosphate groups and Ca2+ cations of ACP, along with H bonds to either protonated or nonprotonated inorganic phosphate groups. The Pser and Ser molecules anchor at their phosphate/amino and carboxy/amino moieties, respectively, leading to an extended molecular conformation across the surface, as opposed to an “upright standing” molecule that would result from the binding of one sole functional group.

Use of Diethanolamine as a Viscous Solvent for Mixture Analysis by Multidimensional Heteronuclear ViscY NMR Experiments.

Diethanolamine/DMSO-d6 as a viscous binary solvent is first reported for the individualization of low-polarity mixture components by multidimensional heteronuclear ViscY NMR experiments under spin diffusion conditions. Solvent viscosity induces the slowing down of molecular tumbling, hence promoting magnetization transfer by dipolar longitudinal cross-relaxation. As a result, all 1H nuclei resonances within the same molecule may correlate in a 2D nuclear Overhauser effect spectroscopy (NOESY) spectrum, giving access to mixture analysis. We offer a new way to analyze mixtures by considering 3D heteronuclear heteronuclear single-quantum coherence-NOESY (HSQC-NOESY) experiments under viscous conditions. We state the individualization of four low-polarity chemical compounds dissolved in the diethanolamine/DMSO-d6 solvent blend using homonuclear selective 1D, 2D 1H-1H NOESY experiments and heteronuclear 1D, 2D 1H-19F heteronuclear Overhauser effect spectroscopy, 2D 1H-19F, 1H-31P HSQC-NOESY, and 3D 1H-19F-1H, 1H-31P-1H HSQC-NOESY experiments by taking profit from spin diffusion.

Application of solid-state NMR techniques for structural characterization of metal-organic frameworks

Solid-state NMR can afford the structural information about the chemical composition, local environment, and spatial coordination at the atomic level, which has been extensively applied to characterize the detailed structure and host-guest interactions in metal-organic frameworks (MOFs). In this review, recent advances for the structural characterizations of MOFs using versatile solid-state NMR techniques were briefly introduced. High-field sensitivity-enhanced solid-state NMR method enabled the direct observation of metal centers in MOFs containing low-γ nuclei. Two-dimensional (2D) homo- and hetero-nuclear correlation MAS NMR experiments provided the spatial proximity among linkers, metal clusters and the introduced guest molecules. Moreover, quantitative measurement of inter-nuclear distances using solid-state NMR provided valuable structural information about the connectivity geometry as well as the host-guest interactions within MOFs. Furthermore, solid-state NMR has exhibited great potential for unraveling the structure property of MOFs containing paramagnetic metal centers.

Two- and Three-Dimensional 13C-17O Heteronuclear Correlation NMR Spectroscopy for Studying Organic and Biological Solids.

We report two- and three-dimensional (2D and 3D) 13C-17O heteronuclear correlation solid-state NMR experiments under magic-angle spinning (MAS) conditions. These experiments utilize the D-RINEPT (Dipolar-mediated Refocused Insensitive Nuclei Enhanced by Polarization Transfer) scheme with symmetry-based SR412 recoupling blocks for coherence transfer between 13C and 17O nuclei. First, a 2D 17O → 13C correlation experiment was performed for the [1-13C,17O]-Gly/Gly·HCl cocrystal and [U-13C, 1-17O]-α/β-d-glucose samples. Second, a 2D 17O → 13C MQ-D-RINEPT correlation experiment where the indirect dimension incorporates the multiple-quantum MAS (MQMAS) scheme was tested for obtaining isotropic 17O resolution with [U-13C, 1-17O]-α/β-d-glucose. Third, a new 3D 17O → 13C → 13C correlation experiment was demonstrated where 17O → 13C and 13C → 13C correlations are achieved by D-RINEPT and DARR (Dipolar Assisted Rotational Resonance) sequences, respectively (thus termed as a 3D D-RINEPT/DARR OCC experiment). This new 3D 17O NMR experiment is implemented with the aim for site-resolved solid-state 17O NMR studies.

Exclusively heteronuclear NMR experiments for the investigation of intrinsically disordered proteins: focusing on proline residues.

NMR represents a key spectroscopic technique that contributes to the emerging field of highly flexible, intrinsically disordered proteins (IDPs) or protein regions (IDRs) that lack a stable three-dimensional structure. A set of exclusively heteronuclear NMR experiments tailored for proline residues, highly abundant in IDPs/IDRs, are presented here. They provide a valuable complement to the widely used approach based on amide proton detection, filling the gap introduced by the lack of amide protons in proline residues within polypeptide chains. The novel experiments have very interesting properties for the investigations of IDPs/IDRs of increasing complexity.

Revealing the Surface Structure of CdSe Nanocrystals by Dynamic Nuclear Polarization-Enhanced 77Se and 113Cd Solid-State NMR Spectroscopy.

Dynamic nuclear polarization (DNP) solid-state NMR (SSNMR) spectroscopy was used to obtain detailed surface structures of zinc blende CdSe nanocrystals (NCs) with plate or spheroidal morphologies which are capped by carboxylic acid ligands. 1D 113Cd and 77Se cross-polarization magic angle spinning (CPMAS) NMR spectra revealed distinct signals from Cd and Se atoms on the surface of the NCs, and those residing in bulk-like environments, below the surface. 113Cd cross-polarization magic-angle-turning (CP-MAT) experiments identified CdSe3O, CdSe2O2, and CdSeO3 Cd coordination environments on the surface of the NCs, where the oxygen atoms are presumably from coordinated carboxylate ligands. The sensitivity gain from DNP enabled natural isotopic abundance 2D homonuclear 113Cd-113Cd and 77Se-77Se and heteronuclear 113Cd-77Se scalar correlation solid-state NMR experiments which revealed the connectivity of the Cd and Se atoms. Importantly, 77Se{113Cd} scalar heteronuclear multiple quantum coherence (J-HMQC) experiments were used to selectively measure one-bond 77Se-113Cd scalar coupling constants (1J(77Se, 113Cd)). With knowledge of 1J(77Se, 113Cd), heteronuclear 77Se{113Cd} spin echo (J-resolved) NMR experiments were used to determine the number of Cd atoms bonded to Se atoms and vice versa. The J-resolved experiments directly confirmed that major Cd and Se surface species have CdSe2O2 and SeCd4 stoichiometries, respectively. Considering the crystal structure of zinc blende CdSe and the similarity of the solid-state NMR data for the platelets and spheroids, we conclude that the surface of the spheroidal CdSe NCs is primarily composed of {100} facets. The methods outlined here will generally be applicable to obtain detailed surface structures of various main group semiconductor nanoparticles.

Stabilization mechanism of amorphous carbamazepine by transglycosylated rutin, a non-polymeric amorphous additive with a high glass transition temperature

α-Glycosyl rutin (Rutin-G), composed of a flavonol skeleton and sugar groups, is a promising non-polymeric additive for stabilizing amorphous drug formulations. In this study, the mechanism of the stabilization of the amorphous state of carbamazepine (CBZ) by Rutin-G was investigated. In comparison with hypromellose (HPMC), which is commonly used as a crystallization inhibitor for amorphous drugs, Rutin-G significantly stabilized amorphous CBZ. Moreover, the dissolution rate and the resultant supersaturation level of CBZ were significantly improved in the CBZ/Rutin-G spray-dried samples (SPDs) owing to the rapid dissolution property of Rutin-G. Differential scanning calorimetry measurement demonstrated a high glass transition temperature (Tg) of 186.4°C corresponding to Rutin-G. The CBZ/Rutin-G SPDs with CBZ weight ratios up to 80% showed single glass transitions, indicating the homogeneity of CBZ and Rutin-G. A solid-state NMR experiment using 13C- and 15N-labeled CBZ demonstrated the interaction between the flavonol skeleton of Rutin-G and the amide group of CBZ. A 1H-13C two-dimensional heteronuclear correlation NMR experiment and quantum mechanical calculations confirmed the presence of a possible hydrogen bond between the amino proton in CBZ and the carbonyl oxygen in the flavonol skeleton of Rutin-G. This specific hydrogen bond could contribute to the strong interaction between CBZ and Rutin-G, resulting in the high stability of amorphous CBZ in the CBZ/Rutin-G SPD. Hence, Rutin-G, a non-polymeric amorphous additive with high Tg, high miscibility with drugs, and rapid and pH-independent dissolution properties could be useful in the preparation of amorphous formulations.

Uncovering selective and active Ga surface sites in gallia-alumina mixed-oxide propane dehydrogenation catalysts by dynamic nuclear polarization surface enhanced NMR spectroscopy.

Gallia–alumina (Ga,Al)2O3(x : y) spinel-type solid solution nanoparticle catalysts for propane dehydrogenation (PDH) were prepared with four nominal Ga : Al atomic ratios (1 : 6, 1 : 3, 3 : 1, 1 : 0) using a colloidal synthesis approach. The structure, coordination environment and distribution of Ga and Al sites in these materials were investigated by X-ray diffraction, X-ray absorption spectroscopy (Ga K-edge) as well as 27Al and 71Ga solid state nuclear magnetic resonance. The surface acidity (Lewis or Brønsted) was probed using infrared spectroscopy with pyridine and 2,6-dimethylpyridine probe molecules, complemented by element-specific insights (Ga or Al) from dynamic nuclear polarization surface enhanced cross-polarization magic angle spinning 15N{27Al} and 15N{71Ga} J coupling mediated heteronuclear multiple quantum correlation NMR experiments using 15N-labelled pyridine as a probe molecule. The latter approach provides unique insights into the nature and relative strength of the surface acid sites as it allows to distinguish contributions from Al and Ga sites to the overall surface acidity of mixed (Ga,Al)2O3 oxides. Notably, we demonstrate that (Ga,Al)2O3 catalysts with a high Al content show a greater relative abundance of four-coordinated Ga sites and a greater relative fraction of weak/medium Ga-based surface Lewis acid sites, which correlates with superior propene selectivity, Ga-based activity, and stability in PDH (due to lower coking). In contrast, (Ga,Al)2O3 catalysts with a lower Al content feature a higher fraction of six-coordinated Ga sites, as well as more abundant Ga-based strong surface Lewis acid sites, which deactivate through coking. Overall, the results show that the relative abundance and strength of Ga-based surface Lewis acid sites can be tuned by optimizing the bulk Ga : Al atomic ratio, thus providing an effective measure for a rational control of the catalyst performance.

Detection of key sites of dimer dissociation and unfolding initiation during activation of acid-stress chaperone HdeA at low pH

HdeA is a small acid-stress chaperone protein with a unique activity profile. At physiological pH, it forms a folded, but inactive, dimer. Below pH 3.0, HdeA unfolds and dissociates into disordered monomers, utilizing exposed hydrophobic patches to bind other unfolded proteins and prevent their irreversible aggregation. In this way, HdeA has a key role in helping pathogenic bacteria survive our acidic stomach and colonize our intestines, facilitating the spread of dysentery. Despite numerous publications on the topic, there remain questions about the mechanism by which HdeA unfolding and activation are triggered. Previous studies usually assessed HdeA unfolding over pH increments that are too far apart to gain fine detail of the process of unfolding and dimer dissociation, and often employed techniques that prevented thorough evaluation of specific regions of the protein. We used a variety of heteronuclear NMR experiments to investigate changes to backbone and side chain structure and dynamics of HdeA at four pHs between 3.0 and 2.0. We found that the long loop in the dimer interface is an early site of initiation of dimer dissociation, and that a molecular “clasp” near the disulfide bond is broken open at low pH as part, or as a trigger, of unfolding; this process also results in the separation of C-terminal helices and exposure of key hydrophobic client binding sites. Our results highlight important regions of HdeA that may have previously been overlooked because they lie too close to the disulfide bond or are thought to be too dynamic in the folded state to influence unfolding processes.

Monitoring the Interaction of α-Synuclein with Calcium Ions through Exclusively Heteronuclear Nuclear Magnetic Resonance Experiments.

Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca2+ concentration jumps associated with the transmission of nerve signals. Novel modules in the primary sequence of α-synuclein optimized for calcium sensing in highly flexible, disordered protein segments are identified.