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Bioactive compounds generally need to cross membranes to arrive at their site of action. The octanol-water partition coefficient (lipophilicity, logP OW) has proven to be an excellent proxy for membrane permeability. In modern drug discovery, logP OW and bioactivity are optimized simultaneously, for which fluorination is one of the relevant strategies. The question arises as to which extent the often subtle logP modifications resulting from different aliphatic fluorine-motif introductions also lead to concomitant membrane permeability changes, given the difference in molecular environment between octanol and (anisotropic) membranes. It was found that for a given compound class, there is excellent correlation between logP OW values with the corresponding membrane molar partitioning coefficients (logK p); a study enabled by novel solid-state 19F NMR MAS methodology using lipid vesicles. Our results show that the factors that cause modulation of octanol-water partition coefficients similarly affect membrane permeability.

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic-angle spinning NMR spectroscopy. Herein, the feasibility of proton-detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected 14N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.

Lipidic bicelles have been widely used for the analysis of integral membrane proteins where their spontaneous alignment in an magnetic field has been exploited for oriented sample solid-state NMR studies. Many of their physical properties however make them well suited to the analysis of membrane proteins by circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD). In this paper we have identified bicelle compositions that permit comparable studies of integral membrane proteins by solid-state NMR, CD, and SRCD; complementary methods that can provide insights into protein structure and the interactions with drugs and other small molecules. Furthermore we have been able to identify bicelle compositions that allow the magnetic alignment of the bicelles at fields routinely available in magnetic CD instruments. This potentially provides a route to the preparation of samples for oriented CD that mitigates many of the issues associated with the preparation of mechanically aligned samples, although we demonstrate that the dynamics present within the system can complicate the analysis of such spectra.

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic‐angle spinning NMR spectroscopy. Herein, the feasibility of proton‐detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected 14N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable., Dynamic nuclear polarization: Proton‐detected 1H/14N dynamic nuclear polarization magic‐angle spinning NMR method provides insights into the structure of biomolecular systems without recourse to isotope labelling. The improvement in sensitivity permits the acquisition of indirectly detected 14N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling.

Magic-angle spinning solid-state NMR is increasingly utilized to study the naturally abundant, spin-1 nucleus 14N, providing insights into the structure and dynamics of biological and organic molecules. In particular, the characterisation of 14N sites using indirect detection has proven useful for complex molecules, where the ‘spy’ nucleus provides enhanced sensitivity and resolution. Here we exploit the sensitivity of proton detection, to indirectly characterise 14N sites using a moderate rf field to generate coherence between the 1H and 14N at moderate and fast-magic-angle spinning frequencies. Efficient numerical simulations have been developed that have allowed us to quantitatively analyse the resulting 14N lineshapes to determine both the size and asymmetry of the quadrupolar interaction. Exploiting only naturally occurring abundant isotopes will aid the analysis of materials with the need to resort to isotope labelling, whilst providing additional insights into the structure and dynamics that the characterisation of the quadrupolar interaction affords.

Magic-angle spinning solid-state NMR is increasingly utilized to study the naturally abundant, spin-1 nucleus 14N, providing insights into the structure and dynamics of biological and organic molecules. In particular, the characterisation of 14N sites using indirect detection has proven useful for complex molecules, where the 'spy' nucleus provides enhanced sensitivity and resolution. Here we exploit the sensitivity of proton detection, to indirectly characterise 14N sites using a moderate rf field to generate coherence between the 1H and 14N at moderate and fast-magic-angle spinning frequencies. Efficient numerical simulations have been developed that have allowed us to quantitatively analyse the resulting 14N lineshapes to determine both the size and asymmetry of the quadrupolar interaction. Exploiting only naturally occurring abundant isotopes will aid the analysis of materials with the need to resort to isotope labelling, whilst providing additional insights into the structure and dynamics that the characterisation of the quadrupolar interaction affords.

Polarization transfer methods are widely adopted for the purpose of correlating different nuclear species as well as to achieve signal enhancement. Polarization transfer from 1H to the 14N overtone transition (Δm = 2) can be achieved using cross polarization methods under magic-angle spinning conditions, where spin locks of the order of several milliseconds can be obtained on common bio-solids (α-glycine and N-acetylvaline). Signal enhancement factors up to 4.4 per scan, can be achieved under favorable conditions, despite MHz-sized quadrupolar interaction. Moreover, we present a detailed theoretical treatment and accurate numerical simulations which are in excellent agreement the unusual experimental matching conditions observed for cross-polarization to 14N overtone.

European Biophysics Journal, 33 (3), ISSN:0175-7571, ISSN:1432-1017

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.

Overtone 14N NMR spectroscopy is a promising route for the direct detection of 14N signals with good spectral resolution. Its application is currently limited, however, by the absence of efficient polarization techniques for overtone signal enhancement and the lack of efficient numerical simulation techniques to aid in both the development of new methods and the analysis and interpretation of experimental data. In this paper we report a novel method for the transfer of polarization from 1H to the 14N overtone using symmetry-based R-sequences that overcome many of the limitations of adiabatic approaches that have worked successfully on static samples. Refinement of these sequences and the analysis of the resulting spectra have been facilitated through the development of an efficient simulation strategy for 14N overtone NMR spectroscopy of spinning samples, using effective Hamiltonians on top of Floquet and Fokker–Planck equations.

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.

In resistive pulse sensing of microRNA biomarkers, selectivity is achieved with polynucleotide-extended DNA probes, with the unzipping of a miRNA−DNA duplex in the nanopore recorded as a resistive current pulse. As the assay sensitivity is determined by the pulse frequency, we investigated the effect of cis/trans electrolyte concentration gradients applied over α-hemolysin nanopores. KCl gradients were found to exponentially increase the pulse frequency, while reducing the preference for 3'-first pore entry of the duplex and accelerating duplex unzipping, all manifestations of an enhanced electrophoretic force. Unlike silicon nitride pores, a counteracting contribution from electro-osmotic flow along the pore wall was not apparent. Significantly, a gradient of 0.5 / 4 M KCl increased the pulse frequency ~60-fold with respect to symmetrical 1 M KCl, while the duplex dwell time in the nanopore remained acceptable for pulse detection and could be extended by LiCl addition. Steeper gradients caused lipid bilayer destabilization and pore instability, limiting the total number of recorded pulses. The 8-fold KCl gradient enabled a linear relationship between pulse frequency and miRNA concentration for the range 0.1−100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for sub-nanomolar miRNA quantification with bilayer-embedded porins.

The hERG channel is a voltage-gated potassium channel found in cardiomyocytes that contributes to the repolarization of the cell membrane following the cardiac action potential, an important step in the regulation of the cardiac cycle. The lipids surrounding K+ channels have been shown to play a key role in their regulation, with anionic lipids shown to alter gating properties. In this study, we investigate how anionic lipids interact with the pore helix of hERG and compare the results with those from Kv1.5, which possesses a pore helix more typical of K+ channels. Circular dichroism studies of the pore helix secondary structure reveal that the presence of the anionic lipid DMPS within the bilayer results in a slight unfolding of the pore helices from both hERG and Kv1.5, albeit to a lesser extent for Kv1.5. In the presence of anionic lipids, the two pore helices exhibit significantly different interactions with the lipid bilayer. We demonstrate that the pore helix from hERG causes significant perturbation to the order in lipid bicelles, which contrasts with only small changes observed for Kv1.5. These observations suggest that the atypical sequence of the pore helix of hERG may play a key role in determining how anionic lipids influence its gating.

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.

5 pags, 3 figs, Oligomeric species populated during the aggregation process of a-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic a-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by a-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons., This research was supported by Parkinson's UK (G-1508 to G.F., M.V., C.M.D., and A.D.); the UK Medical Research Council (MR/N000676/1 to G.F., M.V. C.M.D., and A.D.); the Wellcome Trust (104933/2/14E to kD.): the Leverhulme Trust (RPG-2015-350 to A.D. and RPG-2015-345 to L.Y.); the British Heart Foundation (PG/14/93/31237 to A.D. and PG/11/81/29130 to L.Y.); the UK Biotechnology and Biological Sciences Research Council (BB/M023923/1 to A.D. and BB/G00594X/1 to L.Y.); the Agency of Science, Technology and Research of Singapore (to S.W.C.); the Ministry of Economy, Industry, and Competitiveness of Spain (MINECO RYC-2012-12068 and MINECO/FEDER EU BEU2015-64119-P to N.C.); the Regione Toscana (SUPREMAL to E.C., C.C., and R.C); the University of Florence (Eondi di Ateneo to E.C. and. C.C); and the Centre for Misfolding Diseases of the University of Cambridge. The data supporting the findings of this study are available within the article and supplementary materials.

It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)- and cholesterol (Chol)- driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for ‘rafts’. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Förster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solid-state wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

Bicelles (bilayered micelles) are model membranes used in the study of peptide structure and membrane interactions. They are traditionally made of long- and short-chain phospholipids, usually dimyristoylphosphatidylcholine (D14PC) and dihexanoyl-PC (D6PC). They are attractive membrane mimetics because their composition and planar surface are similar to the native membrane environment. In this work, to improve the solubilization of membrane proteins and allow their study in bicellar systems, D6PC was replaced by detergents from the monoalkylphosphocholine (MAPCHO) family, of which dodecylphosphocholine (12PC) is known for its ability to solubilize membrane proteins. More specifically 12PC, tetradecyl- (14PC), and hexadecyl-PC (16PC) have been employed. To verify the possibility of making bicelles with different hydrophobic thicknesses to better accommodate membrane proteins, D14PC was also replaced by phospholipids with different alkyl chain lengths: dilauroyl-PC (D12PC), dipalmitoyl-PC (D16PC), distearoyl-PC (D18PC), and diarachidoyl-PC (D20PC). Results obtained by 31P solid-state nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) at several lipid-to-detergent molar ratios (q) and temperatures indicate that these new MAPCHO bicelles can be formed under a variety of conditions. The quality of their alignment is similar to that of classical bicelles, and the low critical micelle concentration (CMC) of the surfactants and their miscibility with phospholipids are likely to be advantageous for the reconstitution of membrane proteins.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.

Oriented SRCD spectroscopy is a challenging but revealing tool for investigating the structure of membrane proteins in lipid bilayers. Using the B23 beamtime at Diamond Light Source, Oxfordshire, UK, we have been developing methods for the preparation and measurement of oriented SRCD samples to characterise the structure of integral membrane proteins and their orientation with respect to the lipid bilayer. In the work presented here we outline two methods for the preparation of oriented membrane proteins. The first relies on mechanical orientation obtained by macroscopically aligning bilayers on a Suprasil quartz support. The second exploits magnetic fields to align bicelle membrane mimetics with the membrane director either parallel or perpendicular to the direction of propagation of the light beam. The merits and drawbacks of these two methods for the determination of membrane protein orientation will be discussed.As a model membrane protein for these measurements we are using the transmembrane domain (TMD) of a putative glycosyltransferase Fukutin-I (FkI), whose mislocalization has been linked to the onset of Fukuyama Muscular Dytrophy. The localisation of Fk-I in the late ER/Golgi is thought to be mediated through changes in oligomeric state and structure induced by the surrounding lipids. Using oriented samples for both SRCD and solid-state NMR we are beginning to understand how the distinct lipid composition within the late ER/Golgi may govern the retention of this protein in these compartments.

The N-terminal domain of Fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum/Golgi apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multi-scale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability and orientation of the short 36-residue N-terminal of Fukutin-I (FK1TMD) in lipids of differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when orientated with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, avoiding hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phoshpatidylinositol 4,5-bisphosphate (PIP2) lipids.

The rational development of drugs which target integral membrane proteins relies intimately on our understanding of their interactions with their binding sites. Advances in solid-state NMR methodology coupled to improved expression and purification strategies for membrane proteins now enable us to probe the structure, dynamics, and binding modes of these drugs with ever-increasing resolution. This article seeks to highlight these advances demonstrating how improvements in protein expression and purification can be coupled with recent developments in instrumentation and pulse sequences in magic-angle spinning, static, and oriented sample solid-state NMR to reveal structural and dynamic information of high-affinity drugs/ligands within their binding sites on membrane proteins. Furthermore, we will highlight some of the major experimental considerations associated with performing these studies

Solid-state NMR transitions involving outer energy levels of the spin-1 14N nucleus are immune, to first order in perturbation theory, to the broadening caused by the nuclear quadrupole interaction. The corresponding overtone spectra, when acquired in conjunction with magic-angle sample spinning, result in lines, which are just a few kHz wide, permitting the direct detection of nitrogen compounds without the need for labeling. Despite the success of this technique, “overtone” resonances are still broadened due to indirect, second order effects arising from the large quadrupolar interaction. Here we demonstrate that another order of magnitude in spectral resolution may be gained by using double rotation. This brings the width of the 14N solid-state NMR lines much closer to the region commonly associated with high-resolution solid-state NMR spectroscopy of 15N and demonstrates the improvements in resolution that may be possible through the development of pulsed methodologies to suppress these second order effects.

The morphogenic opiate pentapeptide leucine-enkephalin (lenk) in a hydrated dimyristoylphosphatidylcholine (DMPC) bilayer is studied using NMR spectroscopy and molecular dynamics simulation. Contrary to the frequent assumption that the peptide attains a single fixed conformation in the presence of membranes, we find that the lenk molecule is flexible, switching between specific bent conformations. The constraints to the orientation of the aromatic rings that are identified by the NMR experiment are found by the MD simulation to be related to the depth of the peptide in the bilayer. The motion of the N-H vectors of the peptide bonds with respect to the magnetic field direction as observed by MD largely explain the magnitude of the observed residual dipolar coupling (RDC), which are much reduced over the static (15)N-(1)H coupling. The measured RDCs are nevertheless significantly larger than the predicted ones, possibly due the absence of long-time motions in the simulations. The conformational behavior of lenk at the DMPC surface is compared to that in the aqueous solution, both in the neutral and in the zwitterionic forms.

Exploiting naturally abundant (14)N and (31)P nuclei by high-resolution MAS NMR (magic angle spinning nuclear magnetic resonance) provides a molecular view of the electrostatic potential present at the surface of biological model membranes, the electrostatic charge distribution across the membrane interface, and changes that occur upon peptide association. The spectral resolution in (31)P and (14)N MAS NMR spectra is sufficient to probe directly the negatively charged phosphate and positively charged choline segment of the electrostatic P(-)-O-CH(2)-CH(2)-N(+)(CH(3))(3) headgroup dipole of zwitterionic DMPC (dimyristoylphosphatidylcholine) in mixed-lipid systems. The isotropic shifts report on the size of the potential existing at the phosphate and ammonium group within the lipid headgroup while the chemical shielding anisotropy ((31)P) and anisotropic quadrupolar interaction ((14)N) characterize changes in headgroup orientation in response to surface potential. The (31)P/(14)N isotropic chemical shifts for DMPC show opposing systematic changes in response to changing membrane potential, reflecting the size of the electrostatic potential at opposing ends of the P(-)-N(+) dipole. The orientational response of the DMPC lipid headgroup to electrostatic surface variations is visible in the anisotropic features of (14)N and (31)P NMR spectra. These features are analyzed in terms of a modified "molecular voltmeter" model, with changes in dynamic averaging reflecting the tilt of the C(beta)-N(+)(CH)(3) choline and PO(4)(-) segment. These properties have been exploited to characterize the changes in surface potential upon the binding of nociceptin to negatively charged membranes, a process assumed to proceed its agonistic binding to its opoid G-protein coupled receptor.

It is demonstrated that internuclear distances can be evaluated from rotational-resonance (RR) experiments in uniformly (13)C-labelled compounds. The errors in the obtained distances are less than 10% without the need to know any parameters of the spin system except the isotropic chemical shifts of all spins. We describe the multi-spin system with a simple fictitious spin-1/2 model. The influence of the couplings to the passive spins (J and dipolar coupling) is described by an empirical constant offset from the rotational-resonance condition. Using simulated data for a three-spin system, we show that the two-spin model describes the rotational-resonance transfer curves well as long as none of the passive spins is close to a rotational-resonance condition with one of the active spins. The usability of the two-spin model is demonstrated experimentally using a sample of acetylcholine perchlorate with labelling schemes of various levels of complexity. Doubly-, triply-, and fully labelled compounds lead to strongly varying RR polarization-transfer curves but the evaluated distances using the two-spin model are identical within the expected error limits and coincide with the distance from the X-ray structure. Rotational-resonance distance measurements in fully labelled compounds allow, in particular, the measurement of weak couplings in the presence of strong couplings.

Solid spider dragline silk is well-known for its mechanical properties. Nonetheless a detailed picture of the spinning process is lacking. Here we report NMR studies on the liquid silk within the wide sac of the major ampullate (m.a.) gland from the spider Nephila edulis. The resolution in the NMR spectra is shown to be significantly improved by the application of magic-angle spinning (MAS). From the narrow width of the resonance lines and the chemical shifts observed, it is concluded that the silk protein within the wide sac of the m.a. gland is dynamically disordered throughout the molecule in the sense that each amino acid of a given type senses an identical environment, on average. The NMR data obtained are consistent with an isotropic liquid phase.

Analysis of hydrogen-bond networks in biomolecules can provide a wealth of information into their function, as well as a valuable source of restraints for structure refinement. Nitrogen-14 NMR has the potential to be a valuable tool in the analysis of these networks as subtle differences in H-bonding can result in significant perturbations in the size of the quadrupolar interaction. Here we describe a family of solid-state NMR experiments that permit the characterisation of the quadrupolar interaction at nitrogen-14 sites within biomolecular systems. These measurements have been made possible by combining recent improvements in the indirect detection of nitrogen-14 sites through spin-1/2 “spy” nuclei (Jarvis, 2013) with the enhanced sensitivity afforded by dynamic nuclear polarization and cryogenic temperatures. Under these conditions carbon-13/nitrogen-14 correlation spectra can be obtained in a matter of hours on model microcrystalline systems such as GB3. Combining these data with assignments obtained from carbon-13/nitrogen-15 labelled systems and numerical simulations permits the quantitative analysis of the nitrogen-14 quadrupolar interaction for individual sites with the molecule. These studies reveal that in the microcrystalline GB3, even subtle differences in hydrogen-bonding such as those observed between helical and beta-sheet structures gives rise to changes in quadrupolar interaction of over 200 kHz resulting in excellent nitrogen-14 chemical shift dispersion. We also highlight how these methods can be used in conjunction with proton detected solid-state NMR paving the way to study such H-bond networks in natural materials.Jarvis, J.A., Haies, I.M., et al., An efficient NMR method for the characterisation of 14N sites through indirect 13C detection. Phys Chem Chem Phys, 2013. 15(20): p. 7613-20.

MicroRNAs are emerging as a new class of molecular biomarkers for a range of diseases. Nanopore resistive pulse sensing is a novel technique for the detection and quantification of these short oligonucleotides which, when electrophoretically driven through a nanopore, cause a transient decrease in electrical current. In the presence of a DNA probe complementary to the target miRNA sequence, pore capture and translocation events of miRNA-DNA hybrids are distinguished by the length and depth of the current-block pulse. Because quantification of the miRNA analyte requires a sufficiently high number of these pulses, optimization of the miRNA-DNA capture rate is important. We investigated the translocation rate and dwell time of miRNA-155 annealed to its complementary DNA probe for different electrolyte gradients across a lipid bilayer with an alpha-hemolysin nanopore. The translocation rate was found to increase with higher applied voltages and with steeper electrolyte gradients. The number of current block pulses was two orders of magnitude larger with 0.1 M / 4 M KCl than with 1 M / 1 M KCl electrolyte solutions, while the dwell time was significantly reduced at a bias of 120 mV, but not at higher voltages. By comparing DNA probes with 3′, 5′, or both 3′ and 5′ homonucleotide extensions we also established that the current block detail depends on the probe extension side, which is most likely related to different base pairing at the two miRNA-155 termini. The highest translocation rates were obtained with the dual extended DNA probe but the shortest dwell times with the 3′-only extension. These optimized parameters enable nanopore resistive pulse sensing of miRNA molecules over a larger concentration range.

Model-membrane systems composed of liquid-crystalline bicellar phases can be uniaxially oriented with respect to a magnetic field, thereby facilitating structural and dynamics studies of membrane-associated proteins. Here we quantitatively characterize a method that allows the manipulation of the direction of this uniaxial orientation. Bicelles formed from DMPC/DHPC are examined by (31)P NMR under variable-angle sample-spinning (VAS) conditions, confirming that the orientation of the liquid-crystalline director can be influenced by sample spinning. The director is perpendicular to the rotation axis when Theta (the angle between the sample-spinning axis and the magnetic field direction) is smaller than the magic angle, and is parallel to the rotation axis when Theta is larger than the magic angle. The new (31)P NMR VAS data presented are considerably more sensitive to the orientation of the bicelle than earlier (2)H studies and the analysis of the sideband pattern allows the determination of the orientation of the liquid-crystal director and its variation over the sample, i.e., the mosaic spread. Under VAS, the mosaic spread is small if Theta deviates significantly from the magic angle but becomes very large at the magic angle.

The structural and dynamic analysis of proteins by NMR in the solid-state has typically required extensive isotope labelling. Here we report our progress using the naturally occurring isotopes 1H and 14N to probe the structure and dynamics of biomolecules. Employing indirect-detection methods, we have previously demonstrated the feasibility of characterising the quadrupolar interaction present at the naturally occurring 14N sites within proteins using 13C as a ‘spy’ nucleus (Jarvis, 2013). These methods demonstrated that in well folded proteins, limited dynamic averaging of the quadrupolar interaction occurred and the magnitude of the quadrupolar interaction reflected the nature of the hydrogen bonding experienced by the nitrogen, with differences of up to 200 kHz measured between amides in alpha helices and beta sheets - allowing a detailed analysis of the electronic environment along the protein backbone. We have extended this methodology to exploit protons as a spy nucleus, where significant enhancements in sensitivity are realised. These advances have opened up the possibility of conducting a molecular analysis of unlabelled biomolecules such as ex-vivo or environmental samples. Currently, we are applying these methods to amyloid fibres, to provide insights into their backbone conformation and polymorphism.Jarvis, J.A., Haies, I.M., et al., An efficient NMR method for the characterisation of 14N sites through indirect 13C detection. Phys Chem Chem Phys, 2013. 15(20): p. 7613-20.

Oriented circular dichroism (oCD) spectroscopy is a challenging but revealing technique that is used to ascertain the tilt angle of membrane proteins in oriented lipid bilayers. The preparation of conventional oCD samples, where lipid bilayers are deposited on a support substrate, is a laborious process which can require extensive repetition and optimisation to produce well aligned bilayers with good optical properties. Even then uncertainty over the depth of bilayers deposited and effective path-length results in inconsistency between samples and also within different regions of the same sample.

Over expression of proteins in E. coli frequently results in the production of inclusion bodies. Although β(2) -microglobulin frequently forms fibrillar structures, our studies reveal significant differences between the protein in fibrils and inclusion bodies. This suggests that the formation of fibrils in inclusion bodies is dependent on the propensity of the protein to form fibrillar structures.

Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using 31P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2–400 mM) and q ratios (0.15–2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ≤250 mM and especially at q ∼ 1.5–2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q > 1. Boundaries are presented within which control of the bicelles’ q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.

Integral membrane proteins support a number of important cellular processes playing a key role in the transport of information and material across cellular membranes. Indeed, miss-regulation or miss-localization of these proteins has been linked to several important disease states. It has become abundantly clear that the activity of these proteins and their localization within the cell critically depends on their interactions with other cellular components, including small molecules destined for translocation across the bilayer or involved in cell signalling, lipids present within the lipid bilayer and other integral and soluble proteins. Accordingly, if we are to understand how integral membrane proteins fulfill these important roles it is vital we understand how they interact with their local cellular environment at the molecular level. Solid-state NMR has emerged as a powerful tool to study how integral membrane proteins interact with other cellular partners, facilitating the study of these interactions at the molecular level. Importantly, as we highlight, these processes can now be studied whilst the protein is resident within the lipid bilayer, which provides unique insights into how interactions between the lipid bilayer and integral membrane proteins can play an important role in regulating vital cellular and disease related processes.

Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERGS5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.

Fukutin-I is localised to the endoplasmic reticulum or Golgi apparatus within the cell, where it is believed to function as a glycosyltransferase. Its localisation within the cell is thought to to be mediated by the interaction of its N-terminal transmembrane domain with the lipid bilayers surrounding these compartments, each of which possesses a distinctive lipid composition. However, it remains unclear at the molecular level how the interaction between the transmembrane domains of this protein and the surrounding lipid bilayer drives its retention within these compartments. In this work, we employed chemical cross-linking and fluorescence resonance energy transfer measurements in conjunction with multiscale molecular dynamics simulations to determine the oligomeric state of the protein within dilauroylphosphatidylcholine bilayers to identify interactions between the transmembrane domains and to ascertain any role these interactions may play in protein localisation. Our studies reveal that the N-terminal transmembrane domain of Fukutin-I exists as dimer within dilauroylphosphatidylcholine bilayers and that this interaction is driven by interactions between a characteristic TXXSS motif. Furthermore residues close to the N-terminus that have previously been shown to play a key role in the clustering of lipids are shown to also play a major role in anchoring the protein in the membrane. Electronic supplementary material The online version of this article (doi:10.1007/s00249-011-0773-5) contains supplementary material, which is available to authorized users.

The conformation of the neurotransmitter acetylcholine bound to the fully functional nicotinic acetylcholine receptor embedded in its native membrane environment has been characterized by using frequency-selective recoupling solid-state NMR. Six dipolar couplings among five resolved 13 C-labeled atoms of acetylcholine were measured. Bound acetylcholine adopts a bent conformation characterized with a quaternary ammonium-to-carbonyl distance of 5.1 Å. In this conformation, and with its orientation constrained to that previously determined by us, the acetylcholine could be docked satisfactorily in the agonist pocket of the agonist-bound, but not the agonist-free, crystal structure of a soluble acetylcholine-binding protein from Lymnaea stagnali . The quaternary ammonium group of the acetylcholine was determined to be within 3.9 Å of five aromatic residues and its acetyl group close to residues C187/188 of the principle and residue L112 of the complementary subunit. The observed >C O chemical shift is consistent with H bonding to the nicotinic acetylcholine receptor residues γY116 and δT119 that are homologous to L112 in the soluble acetylcholine-binding protein.

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the gamma-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear alpha-helical conformation with the helix aligned within 15 degrees of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton-proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.

Model-membrane systems composed of liquid-crystalline bicellar phases can be uniaxially oriented with respect to a magnetic field, thereby facilitating structural and dynamics studies of membrane-associated proteins. Here we quantitatively characterize a method that allows the manipulation of the direction of this uniaxial orientation. Bicelles formed from DMPC/DHPC are examined by (31)P NMR under variable-angle sample-spinning (VAS) conditions, confirming that the orientation of the liquid-crystalline director can be influenced by sample spinning. The director is perpendicular to the rotation axis when Theta (the angle between the sample-spinning axis and the magnetic field direction) is smaller than the magic angle, and is parallel to the rotation axis when Theta is larger than the magic angle. The new (31)P NMR VAS data presented are considerably more sensitive to the orientation of the bicelle than earlier (2)H studies and the analysis of the sideband pattern allows the determination of the orientation of the liquid-crystal director and its variation over the sample, i.e., the mosaic spread. Under VAS, the mosaic spread is small if Theta deviates significantly from the magic angle but becomes very large at the magic angle.

Rotational-resonance magic-angle spinning NMR experiments are frequently used to measure dipolar couplings and to determine internuclear distances. So far most measurements were performed on samples containing isolated spin pairs. Thus, extensive structure elucidation, for example in biomolecules, requires the preparation of a whole set of doubly labeled samples. Here, we describe the analysis of the rotational-resonance polarization-exchange curves obtained from a single, uniformly labeled sample. It is shown experimentally that, at a magnetic field of 14.09 T, the rotational-resonance conditions in uniformly (13)C-labeled threonine are sufficiently narrow to permit the measurement of five distances between the four carbon spins with an accuracy of better than 10%. The polarization-exchange curves are analyzed using a modified two-spin model consisting of the two active spins. The modified model includes an additional offset in the final polarization, which comes from the coupling to the additional, passive, spins. The validity of this approach is experimentally verified for uniformly (13)C-labeled threonine. The broader applicability of such a model is demonstrated by numerical simulations which quantify the errors as a function of the most relevant parameters in the spin system.