Velocity selective spin labeling using parallel transmission.

PURPOSE: Ultra-high field (UHF) provides improved SNR which greatly benefits SNR starved imaging techniques such as perfusion imaging. However, transmit field (B1 + ) inhomogeneities commonly observed at UHF hinders the excitation uniformity. Here we show how replacing standard excitation pulses with parallel transmit pulses can improve efficiency of velocity selective labeling. METHODS: The standard tip-down and tip-up excitation pulses found in a velocity selective preparation module were replaced with tailored non-selective kT -points pulse solutions. Bloch simulations and experimental validation on a custom-built flow phantom and in vivo was performed to evaluate different pulse configurations in circularly polarized mode (CP-mode) and parallel transmit (pTx) mode. RESULTS: Tailored pTx pulses significantly improved velocity selective labeling fidelity and signal uniformity. The transverse magnetization normalized RMS error was reduced from 0.489 to 0.047 when compared to standard rectangular pulses played in CP-mode. Simulations showed that manipulation of time symmetry in the tailored pTx pulses is vital in minimizing residual magnetization. In addition, in vivo experiments achieved a 44% lower RF power output and a shorter pulse duration when compared to using adiabatic pulses in CP-mode. CONCLUSION: Using tailored pTx pulses for excitation within a velocity selective labeling preparation mitigated transmit field artifacts and improved SNR and contrast fidelity. The improvement in labeling efficiency highlights the potential of using pTx to improve robustness and accessibility of flow-based sequences such as velocity selective spin labeling at ultra-high field.

Exploring label dynamics of velocity-selective arterial spin labeling in the kidney.

PURPOSE: Velocity-selective arterial spin labeling (VSASL) has been proposed for renal perfusion imaging to mitigate planning challenges and effects of arterial transit time (ATT) uncertainties. In VSASL, label generation may shift in the vascular tree as a function of cutoff velocity. Here, we investigate label dynamics and especially the ATT of renal VSASL and compared it with a spatially selective pulsed arterial spin labeling technique, flow alternating inversion recovery (FAIR). METHODS: Arterial spin labeling data were acquired in 7 subjects, using free-breathing dual VSASL and FAIR with five postlabeling delays: 400, 800, 1200, 2000, and 2600 ms. The VSASL measurements were acquired with cutoff velocities of 5, 10, and 15 cm/s, with anterior-posterior velocity-encoding direction. Cortical perfusion-weighted signal, temporal SNR, quantified renal blood flow, and arterial transit time were reported. RESULTS: In contrast to FAIR, renal VSASL already showed fairly high signal at the earliest postlabeling delays, for all cutoff velocities. The highest VSASL signal and temporal SNR was obtained with a cutoff velocity of 10 cm/s at postlabeling delay = 800 ms, which was earlier than for FAIR at 1200 ms. Fitted ATT on VSASL was ≤ 0 ms, indicating ATT insensitivity, which was shorter than for FAIR (189 ± 79 ms, P < .05). Finally, the average cortical renal blood flow measured with cutoff velocities of 5 cm/s (398 ± 84 mL/min/100 g) and 10 cm/s (472 ± 160 mL/min/100 g) were similar to renal blood flow measured with FAIR (441 ± 84 mL/min/100 g) (P > .05) with good correlations on subject level. CONCLUSION: Velocity-selective arterial spin labeling in the kidney reduces ATT sensitivity compared with the recommended pulsed arterial spin labeling method, as well as if cutoff velocity is increased to reduce spurious labeling due to motion. Thus, VSASL has potential as a method for time-efficient, single-time-point, free-breathing renal perfusion measurements, despite lower tSNR than FAIR.

DNA Tiling Enables Precise Acylation-Based Labeling and Control of mRNA.

Methods for the site-selective labeling of long, native RNAs are needed for studying mRNA biology and future therapies. Current approaches involve engineering RNA sequences, which may alter folding, or are limited to specific sequences or bases. Here, we describe a versatile strategy for mRNA conjugation via a novel DNA-tiling approach. The method, TRAIL, exploits a pool of "protector" oligodeoxynucleotides to hybridize and block the mRNA, combined with an "inducer" DNA that extrudes a reactive RNA loop for acylation at a predetermined site. Using TRAIL, an azido-acylimidazole reagent was employed for labeling and controlling RNA for multiple applications in vitro and in cells, including analysis of RNA-binding proteins, imaging mRNA in cells, and analysis and control of translation. The TRAIL approach offers an efficient and accessible way to label and manipulate RNAs of virtually any length or origin without altering native sequence.

Amino-acid selective isotope labeling enables simultaneous overlapping signal decomposition and information extraction from NMR spectra.

Signal overlapping is a major bottleneck for protein NMR analysis. We propose a new method, stable-isotope-assisted parameter extraction (SiPex), to resolve overlapping signals by a combination of amino-acid selective isotope labeling (AASIL) and tensor decomposition. The basic idea of Sipex is that overlapping signals can be decomposed with the help of intensity patterns derived from quantitative fractional AASIL, which also provides amino-acid information. In SiPex, spectra for protein characterization, such as 15N relaxation measurements, are assembled with those for amino-acid information to form a four-order tensor, where the intensity patterns from AASIL contribute to high decomposition performance even if the signals share similar chemical shift values or characterization profiles, such as relaxation curves. The loading vectors of each decomposed component, corresponding to an amide group, represent both the amino-acid and relaxation information. This information link provides an alternative protein analysis method that does not require "assignments" in a general sense; i.e., chemical shift determinations, since the amino-acid information for some of the residues allows unambiguous assignment according to the dual selective labeling. SiPex can also decompose signals in time-domain raw data without Fourier transform, even in non-uniformly sampled data without spectral reconstruction. These features of SiPex should expand biological NMR applications by overcoming their overlapping and assignment problems.

Influence of labeling parameters and respiratory motion on velocity-selective arterial spin labeling for renal perfusion imaging.

PURPOSE: Arterial transit time uncertainties and challenges during planning are potential issues for renal perfusion measurement using spatially selective arterial spin labeling techniques. To mitigate these potential issues, a spatially non-selective technique, such as velocity-selective arterial spin labeling (VSASL), could be an alternative. This article explores the influence of VSASL sequence parameters and respiratory induced motion on VS-label generation. METHODS: VSASL data were acquired in human subjects (n = 15), with both single and dual labeling, during paced-breathing, while essential sequence parameters were systematically varied; (1) cutoff velocity, (2) labeling gradient orientation and (3) post-labeling delay (PLD). Pseudo-continuous ASL was acquired as a spatially selective reference. In an additional free-breathing single VSASL experiment (n = 9) we investigated respiratory motion influence on VS-labeling. Absolute renal blood flow (RBF), perfusion weighted signal (PWS), and temporal signal-to-noise ratio (tSNR) were determined. RESULTS: (1) With decreasing cutoff velocity, tSNR and PWS increased. However, undesired tissue labeling occurred at low cutoff velocities (≤ 5.4 cm/s). (2) Labeling gradient orientation had little effect on tSNR and PWS. (3) For single VSASL high signal appeared in the kidney pedicle at PLD < 800 ms, and tSNR and PWS decreased with increasing PLD. For dual VSASL, maximum tSNR occurred at PLD = 1200 ms. Average cortical RBF measured with dual VSASL (264 ± 34 mL/min/100 g) at a cutoff velocity of 5.4 cm/s, and feet-head labeling was slightly lower than with pseudo-continuous ASL (283 ± 55 mL/min/100 g). CONCLUSION: With well-chosen sequence parameters, tissue labeling induced by respiratory motion can be minimized, allowing to obtain good quality RBF maps using planning-free labeling with dual VSASL.

Improved velocity-selective labeling pulses for myocardial ASL.

PURPOSE: To develop and evaluate an improved velocity-selective (VS) labeling pulse for myocardial arterial spin labeling (ASL) perfusion imaging that addresses two limitations of current pulses: (1) spurious labeling of moving myocardium and (2) low labeling efficiency. METHODS: The proposed myocardial VSASL labeling pulse is designed using a Fourier Transform based Velocity-Selective labeling pulse train. The pulse utilizes bipolar velocity-encoding gradients, a 9-tap velocity-encoding envelope, and double-refocusing pulses with Malcolm Levitt phase cycling. Amplitudes of the velocity-encoding envelope were optimized to minimize the labeling of myocardial velocities during stable diastole (±2-3 cm/s) and maximize the labeling of coronary velocities (10-130 cm/s during rest/stress or 10-70 cm/s during rest). Myocardial ASL experiments were performed in seven healthy subjects using the previously developed VS-ASL protocol by Jao et al with the two proposed VS pulses and original VS pulse. Myocardial ASL experiments were also performed using FAIR ASL. Myocardial perfusion and physiological noise (PN) were evaluated and compared. RESULTS: Bloch simulations of the first and second proposed pulses show <2% labeling over ±3 cm/s and ±2 cm/s, respectively. Bloch simulations also show the mean labeling efficiency of arterial blood is 1.23 over the relevant coronary arterial ranges. In-vivo VSASL experiments show the proposed pulses provided comparable measurements to FAIR ASL and reduced TSNR in 5 of 7 subjects compared to the original VS pulse. CONCLUSION: We demonstrate an improved VS labeling pulse specifically for myocardial ASL perfusion imaging to reduce spurious labeling of moving myocardium and PN.

Optimization of the spatial modulation function of vessel-encoded pseudo-continuous arterial spin labeling and its application to dynamic angiography.

PURPOSE: In vessel-encoded pseudo-continuous arterial spin labeling (ve-pCASL), vessel-selective labeling is achieved by modulation of the inversion efficiency across space. However, the spatial transition between the labeling and control conditions is rather gradual, which can cause partial labeling of vessels, reducing SNR-efficiency and necessitating complex postprocessing to decode the vessel-selective signals. The purpose of this study is to optimize the pCASL labeling parameters to obtain a sharper spatial inversion profile of the labeling and thereby minimizing the risk of partial labeling of untargeted arteries. METHODS: Bloch simulations were performed to investigate how the inversion profile was influenced by the pCASL labeling parameters: the maximum (Gmax ) and mean (Gmean ) labeling gradient were varied for ve-pCASL with unipolar and bipolar gradients. The findings in the simulation study were subsequently confirmed in an in vivo volunteer study. Moreover, conventional and optimized settings were compared for 4D-MRA using four-cycle Hadamard ve-pCASL; the visualization of arteries and the presence of the partial labeling were assessed by an expert observer. RESULTS: When using unipolar gradient, lower Gmean resulted in a steeper spatial transition, whereas the width of the control region was broader for higher Gmax . The in vivo study confirmed these findings. When using bipolar gradients, the control region was always very narrow. Qualitative comparison of the 4D-MRA demonstrated lower occurrence of partial labeling when using the optimized gradient parameters. CONCLUSION: The shape of the ve-pCASL inversion profile can be optimized by changing Gmean and Gmax to reduce partial labeling of untargeted arteries.

Demonstration of velocity selective myocardial arterial spin labeling perfusion imaging in humans.

PURPOSE: Transit delay is a potential source of error in cardiac arterial spin-labeled (ASL) in heart failure or with collateral circulation. This study demonstrates the feasibility of using transit delay insensitive velocity selective ASL and compares its performance with flow-sensitive alternating inversion recovery (FAIR) ASL. METHODS: Velocity selective labeling was achieved using an adiabatic BIR8 preparation. FAIR and velocity-selective ASL (VSASL) with various velocity cutoffs (VC = 10-40 cm/s) and labeling directions (anterior-posterior X, lateral-septal Y, and apical-basal Z) were carried out in 10 healthy volunteers (1F/9M age 23-30 y). Myocardial blood flow (MBF) and temporal signal-to-noise (TSNR) were measured. RESULTS: VSASL sensitivity to perfusion decreased with increasing VC . At low VC (<5 cm/s), spurious labeling of myocardium occurs and overestimates MBF. MBF measured with FAIR (1.12 ± 0.26 ml/g/min) and VASL (1.26 ± 0.27 ml/g/min) at VC of 10 cm/s in Z were comparable (TOST with difference of 0.30 ml/g/min, P = 0.049). TSNR was 2.8 times larger using FAIR (13.62 ± 5.25) than in VSASL (4.87 ± 1.58). VSASL was insensitive to perfusion in the Y direction. X and Z performed similarly with TSNR of 4.17 ± 2.32 and 3.97 ± 0.56, respectively. CONCLUSION: VSASL is a promising alternative to FAIR ASL in the heart and is well suited for scenarios when transit delays are long. Magn Reson Med 80:272-278, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Advances in stable isotope assisted labeling strategies with information science.

Stable-isotope (SI) labeling of proteins is an essential technique to investigate their structures, interactions or dynamics by nuclear magnetic resonance (NMR) spectroscopy. The assignment of the main-chain signals, which is the fundamental first step in these analyses, is usually achieved by a sequential assignment method based on triple resonance experiments. Independently of the triple resonance experiment-based sequential assignment, amino acid-selective SI labeling is beneficial for discriminating the amino acid type of each signal; therefore, it is especially useful for the signal assignment of difficult targets. Various combinatorial selective labeling schemes have been developed as more sophisticated labeling strategies. In these strategies, amino acids are represented by combinations of SI labeled samples, rather than simply assigning one amino acid to one SI labeled sample as in the case of conventional amino acid-selective labeling. These strategies have proven to be useful for NMR analyses of difficult proteins, such as those in large complex systems, in living cells, attached or integrated into membranes, or with poor solubility. In this review, recent advances in stable isotope assisted labeling strategies will be discussed.

Facilitating unambiguous NMR assignments and enabling higher probe density through selective labeling of all methyl containing amino acids.

The deuteration of proteins and selective labeling of side chain methyl groups has greatly enhanced the molecular weight range of proteins and protein complexes which can be studied using solution NMR spectroscopy. Protocols for the selective labeling of all six methyl group containing amino acids individually are available, however to date, only a maximum of five amino acids have been labeled simultaneously. Here, we describe a new methodology for the simultaneous, selective labeling of all six methyl containing amino acids using the 115 kDa homohexameric enzyme CoaD from E. coli as a model system. The utility of the labeling protocol is demonstrated by efficiently and unambiguously assigning all methyl groups in the enzymatic active site using a single 4D (13)C-resolved HMQC-NOESY-HMQC experiment, in conjunction with a crystal structure. Furthermore, the six fold labeled protein was employed to characterize the interaction between the substrate analogue (R)-pantetheine and CoaD by chemical shift perturbations, demonstrating the benefit of the increased probe density.

Stable isotope labeling strategy based on coding theory.

We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as "encoding and decoding" processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells.

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.

NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.

Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism.

Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

Imitation-mussel fluorescent silicon quantum dots for selective labeling and imaging of bacteria and biofilms.

Selective labeling of distinct bacteria and biofilm is poised for the fundamental understanding of bacterial activities, interactions, and coupled phenomena occurring at the microscale. However, a simple and effective way to achieve selective bacterial labeling is still lacking. Herein, we report a fluorescence probe with core-shell nanostructure that has polydopamine (PDA) coating on the surface of fluorescent silicon quantum dots (SiQDs@PDA). The surface of the SiQDs@PDA can be functionalized by various molecules (2-mercaptoethylamine hydrochloride, PEG, d-alanine, glucose amide) through different strategies (Michael addition, π-π interaction, and ion-ion interaction). Importantly, the d-alanine (D-Ala)- and gluconamide (Glc)-functionalized SiQDs@PDA fluorescence probes are capable of selectively labeling gram-positive and gram-negative bacteria, as well as their biofilms. The excellent performance in universal functionalization and selective labeling and imaging of bacteria and their biofilms demonstrate that SiQDs@PDA are a promising fluorescence tool in microbe research.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41 kDa catalytic domain of USP7 harbors the active site of the enzyme. Here we present a nearly complete (93%) NMR resonance assignment of isoleucine, leucine and valine (ILV) side-chains of the USP7 catalytic domain along with a refined nearly complete (93%) assignment of its backbone resonances. The reported ILV methyl group assignment will facilitate further NMR investigations of structure, interactions and conformational dynamics of the USP7 enzyme.

Organelle-Selective Labeling of Choline-Containing Phospholipids (CPLs) and Real-Time Imaging in Living Cells.

Choline-containing phospholipids (CPLs), including phosphatidylcholine (PC) and sphingomyelin (SM), are the major components of mammalian cell membranes and play critical roles during a variety of cellular processes. However, intracellular dynamics of CPLs is poorly understood due to a lack of methods to trace CPL trafficking at organelle resolution. Here, we describe protocols that make it possible to fluorescently label CPLs at the targeted organelles and to monitor their movement within living cells using confocal microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: ER-Golgi-selective labeling of azide-tagged CPLs for confocal imaging Basic Protocol 2: Mitochondria-selective labeling of azide-tagged CPLs for confocal imaging.

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2.

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.

Optimal Isotope Labeling of Aromatic Amino Acid Side Chains for NMR Studies of Protein Dynamics.

Aromatic side chains in proteins are often directly evolved in stabilizing the hydrophobic core, protein binding, or enzymatic activity. They are also responsible for specific local dynamic processes, such as histidine tautomerization or ring flips. Despite their importance, they are often not targeted directly by NMR spectroscopy, because of spectroscopic complications and challenges. This chapter addresses state-of-the-art site-selective 13C-labeling methods for aromatic side chains, and describes how they solve several of the spectroscopic issues. A special emphasis is put on thereby enabled protein dynamics experiments of aromatic side chains.

Towards complete polypeptide backbone NH assignment via combinatorial labeling.

Combinatorial selective isotope labeling is a valuable tool to facilitate polypeptide backbone resonance assignment in cases of low sensitivity or extensive chemical shift degeneracy. It involves recording of 15N-HSQC and 2D HN-projections of triple-resonance spectra on a limited set of samples containing different combinations of labeled and unlabeled amino acid types. Using labeling schemes in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes - individually as well as simultaneously - usually yields abundant amino-acid type information of consecutive residues i and i - 1. Although this results in a large number of anchor points that can be used in the sequential assignment process, for most amide signals the exact positioning of the corresponding residue the polypeptide sequence still relies on matching intra- and interresidual 13C chemical shifts obtained from 3D spectra. An obvious way to obtain more sequence-specific assignments directly with combinatorial labeling would be to increase the number of samples. This is, however, undesirable because of increased sample preparation efforts and costs. Irrespective of the number of samples, unambiguous assignments cannot be accomplished for i - 1/i pairs that are not unique in the sequence. Here we show that the ambiguity for non-unique pairs can be resolved by including information about the labeling state of residues i + 1 and i - 2. Application to a 35-residue peptide resulted in complete assignments of all detectable signals in the 15N HSQC which, due to its repetitive sequence and 13C chemical shift degeneracies, was difficult to achieve by other means. For a medium-sized protein (165 residues, rotational correlation time 8.2 ns) the improved protocol allowed the extent of backbone amide assignment to be expanded to 88% solely using a suite of 2D 1H-15N correlated spectra.

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel.

Voltage-gated sodium (NaV) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of NaV1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of NaV1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine micelles. To speed up the assignment of backbone resonances, five selectively 13C,15N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ∼50% of the backbone. Obtained NMR and electron paramagnetic resonance data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps-ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein-protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding ß-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one NaV molecule.