Reducing seed dependent variability of non-uniformly sampled multidimensional NMR data

The application of NMR spectroscopy to study the structure, dynamics and function of macromolecules requires the acquisition of several multidimensional spectra. The one-dimensional NMR time-response from the spectrometer is extended to additional dimensions by introducing incremented delays in the experiment that cause oscillation of the signal along “indirect” dimensions. For a given dimension the delay is incremented at twice the rate of the maximum frequency (Nyquist rate). To achieve high-resolution requires acquisition of long data records sampled at the Nyquist rate. This is typically a prohibitive step due to time constraints, resulting in sub-optimal data records to the detriment of subsequent analyses. The multidimensional NMR spectrum itself is typically sparse, and it has been shown that in such cases it is possible to use non-Fourier methods to reconstruct a high-resolution multidimensional spectrum from a random subset of non-uniformly sampled (NUS) data. For a given acquisition time, NUS has the potential to improve the sensitivity and resolution of a multidimensional spectrum, compared to traditional uniform sampling. The improvements in sensitivity and/or resolution achieved by NUS are heavily dependent on the distribution of points in the random subset acquired. Typically, random points are selected from a probability density function (PDF) weighted according to the NMR signal envelope. In extreme cases as little as 1% of the data is subsampled. The heavy under-sampling can result in poor reproducibility, i.e. when two experiments are carried out where the same number of random samples is selected from the same PDF but using different random seeds. Here, a jittered sampling approach is introduced that is shown to improve random seed dependent reproducibility of multidimensional spectra generated from NUS data, compared to commonly applied NUS methods. It is shown that this is achieved due to the low variability of the inherent sensitivity of the random subset chosen from a given PDF. Finally, it is demonstrated that metrics used to find optimal NUS distributions are heavily dependent on the inherent sensitivity of the random subset, and such optimisation is therefore less critical when using the proposed sampling scheme

Nonuniform sampling and non-Fourier signal processing methods in multidimensional NMR

Beginning with the introduction of Fourier Transform NMR by Ernst and Anderson in 1966, time domain measurement of the impulse response (the free induction decay, FID) consisted of sampling the signal at a series of discrete intervals. For compatibility with the discrete Fourier transform (DFT), the intervals are kept uniform, and the Nyquist theorem dictates the largest value of the interval sufficient to avoid aliasing. With the proposal by Jeener of parametric sampling along an indirect time dimension, extension to multidimensional experiments employed the same sampling techniques used in one dimension, similarly subject to the Nyquist condition and suitable for processing via the discrete Fourier transform. The challenges of obtaining high-resolution spectral estimates from short data records using the DFT were already well understood, however. Despite techniques such as linear prediction extrapolation, the achievable resolution in the indirect dimensions is limited by practical constraints on measuring time. The advent of non-Fourier methods of spectrum analysis capable of processing nonuniformly sampled data has led to an explosion in the development of novel sampling strategies that avoid the limits on resolution and measurement time imposed by uniform sampling. The first part of this review discusses the many approaches to data sampling in multidimensional NMR, the second part highlights commonly used methods for signal processing of such data, and the review concludes with a discussion of other approaches to speeding up data acquisition in NMR

A complicated complex: ion channels, voltage sensing, cell membranes and peptide inhibitors

Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Nas) have attracted particular attention as potential analgesic targets. Nas, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na1.7) to controlling electrical signalling in cardiac function (Na1.5). Understanding the structural basis of Na function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Nas by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays

Mapping the molecular surface of the analgesic NaV1.7-selective peptide Pn3a reveals residues essential for membrane and channel interactions

Compelling human genetic studies have identified the voltage-gated sodium channel NaV1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider venom-derived peptide µtheraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of NaV1.7, however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed seventeen Pn3a analogues and determined their activity at hNaV1.7 using patchclamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNaV1.7. Removal of hydrophobic residues Y4, Y27 and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at NaV1.7 (20-fold), whilst maintaining >100-fold selectivity over the major off-targets NaV1.4, NaV1.5 and NaV1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of post-surgical pain at doses 3-fold lower than wild-type Pn3a, without causing motor adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic NaV1.7 inhibitors for the development of more efficacious and safer analgesics but also to further investigate the involvement of NaV1.7 in pain

Two proteins for the price of one: structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1

Seed storage proteins are both an important source of nutrition for humans and essential for seedling establishment. Interestingly, unusual napin-type 2S seed storage albumin precursors in sunflowers contain a sequence that is released as a macrocyclic peptide during post-translational processing. The mechanism by which such peptides emerge from linear precursor proteins has received increased attention; however, the structural characterization of intact precursor proteins has been limited. Here, we report the 3D NMR structure of the Helianthus annuus PawS1 (preproalbumin with sunflower trypsin inhibitor-1) and provide new insights into the processing of this remarkable dual-destiny protein. In seeds, PawS1 is matured by asparaginyl endopeptidases (AEPs) into the cyclic peptide SFTI-1 (sunflower trypsin inhibitor-1) and a heterodimeric 2S albumin. The structure of PawS1 revealed that SFTI-1 and the albumin are independently folded into well-defined domains separated by a flexible linker. PawS1 was cleaved in vitro with recombinant sunflower HaAEP1 and in situ using a sunflower seed extract in a way that resembled the expected in vivo cleavages. Recombinant HaAEP1 cleaved PawS1 at multiple positions, and in situ, its flexible linker was removed, yielding fully mature heterodimeric albumin. Liberation and cyclization of SFTI-1, however, was inefficient, suggesting that specific seed conditions or components may be required for in vivo biosynthesis of SFTI-1. In summary, this study has revealed the 3D structure of a macrocyclic precursor protein and provided important mechanistic insights into the maturation of sunflower proalbumins into an albumin and a macrocyclic peptide

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large beta-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its `depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker

Nonuniform sampling and maximum entropy reconstruction in multidimensional NMR

NMR spectroscopy is one of the most powerful and versatile analytic tools available to chemists. The discrete Fourier transform (DFT) played a seminal role in the development of modern NMR, including the multidimensional methods that are essential for characterizing complex biomolecules. However, it suffers from well-known limitations: chiefly the difficulty in obtaining high-resolution spectral estimates from short data records. Because the time required to perform an experiment is proportional to the number of data samples, this problem imposes a sampling burden for multidimensional NMR experiments. At high magnetic field, where spectral dispersion is greatest, the problem becomes particularly acute. Consequently multidimensional NMR experiments that rely on the DFT must either sacrifice resolution in order to be completed in reasonable time or use inordinate amounts of time to achieve the potential resolution afforded by high-field magnets.Maximum entropy (MaxEnt) reconstruction is a non-Fourier method of spectrum analysis that can provide high-resolution spectral estimates from short data records. It can also be used with nonuniformly sampled data sets. Since resolution is substantially determined by the largest evolution time sampled, nonuniform sampling enables high resolution while avoiding the need to uniformly sample at large numbers of evolution times. The Nyquist sampling theorem does not apply to nonuniformly sampled data, and artifacts that occur with the use of nonuniform sampling can be viewed as frequency-aliased signals. Strategies for suppressing nonuniform sampling artifacts include the careful design of the sampling scheme and special methods for computing the spectrum. Researchers now routinely report that they can complete an N-dimensional NMR experiment 3 times faster (a 3D experiment in one ninth of the time). As a result, high-resolution three- and four-dimensional experiments that were prohibitively time consuming are now practical. Conversely, tailored sampling in the indirect dimensions has led to improved sensitivity.Further advances in nonuniform sampling strategies could enable further reductions in sampling requirements for high resolution NMR spectra, and the combination of these strategies with robust non-Fourier methods of spectrum analysis (such as MaxEnt) represent a profound change in the way researchers conduct multidimensional experiments. The potential benefits will enable more advanced applications of multidimensional NMR spectroscopy to study biological macromolecules, metabolomics, natural products, dynamic systems, and other areas where resolution, sensitivity, or experiment time are limiting. Just as the development of multidimensional NMR methods presaged multidimensional methods in other areas of spectroscopy, we anticipate that nonuniform sampling approaches will find applications in other forms of spectroscopy