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A functionally active analogue of neurotensin, neurotensin(8–13), has been observed whilst bound to the agonist-binding site of the rat neurotensin receptor by nuclear magnetic resonance (NMR). Through the application of slow magic angle sample spinning and high-power proton decoupling, sufficient resolution and sensitivity were obtained in the carbon-13 spectrum to allow an assignment of many of the side chain resonances arising from uniformly carbon-13/nitrogen-15-labelled neurotensin(8–13) whilst bound to the neurotensin receptor. Significant perturbations in carbon-13 chemical shift were observed upon the binding of the neurotensin(8–13) to the receptor. Most importantly significant shifts were observed in both the carboxy terminus and tyrosine side chain of the neurotensin(8–13), suggesting that these sites are important in the interaction of the neurotensin with the agonist-binding site on the neurotensin receptor. Conversely, no perturbations were observed for the carbon-13 sites within the guanidinium groups of the arginine side chains, indicating little interaction with the receptor-binding site, or a shielding of the local environment by the surrounding nitrogen atoms. These NMR observations lend further support to previous structure–activity studies, site-directed mutagenesis and modelling studies of the agonist-binding site of the neurotensin receptor, from which the same specific residues for which NMR perturbations were observed are important for neurotensin receptor activation by neurotensin.

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N + (CD 3 ) 3 -BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42° in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

The neurotensin receptor is a member of the G-protein coupled receptor (GPCR) family of transmembrane proteins that is activated upon the binding of the basic tridecapeptide agonist, neurotensin, to the extracellular surfaces of cells. The neurotensin receptor is found widely in both the central nervous system and the periphery. In the periphery, it stimulates smooth muscle contraction [1,2]. In the central nervous system, it mediates a variety of activities including antinociception, hypothermia and increased locomoter activity [3–5]. These effects are probably mediated through the regulation of the mesolimbic and negrostriatal dopamine pathways [6,7]. As a result, the pharmacological action of the neurotensin is similar to that observed for dopamine, where compounds function as antipsychotics [6,7], and intervention may provide useful insights for the development of treatments for conditions such as schizophrenia [8] and Parkinson’s disease [9].

Alzheimer’s disease (AD) is a chronic dementia, affecting an increasingly large number of old people worldwide [1–3]. AD together with mature onset diabetes and prion-transmissible spongiform encephalopathies, belongs to a category of amyloid diseases, which are all categorized by an abnormal folding of a normally soluble protein into neurotoxic aggregated structures [3–5]. The key event in AD is the metabolism of amyloid precursor protein to amyloid-β-peptide (Aβ) and the subsequent deposition of Aβ as plaques in the brains of patients. This 39–42 amino acid peptide has been linked to the apoptosis of neuronal cells, and its neurotoxicity seems to be associated with its ability to convert from a non-toxic monomeric form into toxic aggregates [5–7]. However the cellular mechanism involved in mediating the toxic effect of Aβ peptide remains unclear [6–11]; and also the process of transformation into insoluble, neurotoxic peptide aggregates. Due to the complexity and dependence of this process on physiological parameters, various models for fibril formation are studied at present including aggregation in solution [7,8], lipid-mediated aggregation of Aβ in contact with cell membrane surfaces [10–13], and formation of transmembrane ion channel-like structures in neuronal membranes [9,14,15]. Structural and biophysical studies of the self-assembly of Aβ-peptide into fibrillar structures found this process strongly dependent on the physical conditions [5,6–8,16]. While earlier studies proposed antiparallel-β-sheet structures for the amyloid fibrils [6], more recent work indicates an in register, parallel organization of β-sheets propagating and twisting along the fibrillar axis [5,17]. However, there is growing evidence that the toxic agent is not the mature fibrils themselves, but rather their precursor forms called diffusible “protofibrils” [3,4,8].

An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (

Acetylcholine, the agonist for the nicotinic acetylcholine receptor, has been observed directly when bound specifically to its binding site in the fully functional receptor-enriched membranes from Torpedo nobiliana. High-resolution solid-state, magic angle spinning 13C NMR methods have been used to observe selectively N+(13CH3)3 acetylcholine bound in as few as 20 nmol of receptor binding sites, against a background of natural abundance membrane resonances and excess acetylcholine in free solution. The specificity of the binding has been demonstrated to be pharmacologically significant through the use of the competitive inhibitor alpha bungarotoxin which selectively displaces and prevents binding of acetylcholine to the membrane-bound receptor. The chemical shift assigned to N+(13CH3)3 acetylcholine in solution and crystalline solid is 53.9 +/- 0.04 ppm, and it changes by 1.6 ppm (p < 0.05) for agonist when bound specifically in the receptor binding site. Through the use of computer simulations of chemical shifts carried out on acetylcholine bound to the acetylcholinesterase, we propose that the cause for this change is the presence of aromatic side chains lining the receptor binding site. It is suggested that the binding of acetylcholine to the nicotinic acetylcholine receptor is mediated primarily through the interaction of the quaternary ammonium group of the acetylcholine with the pi bonded systems in the aromatic side chains. Longitudinal relaxation time measurements show that the residency time for the acetylcholine observed in DDCP experiments is long (> 200 ms) with respect to the longitudinal relaxation time of other assignable resonances within the spectrum from the lipid and protein and confirms that the acetylcholine is protein-associated, and not free in solution or nonspecifically bound.

Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (2H, 13C, 19F, 15N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 A). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.

One approach for obtaining high-resolution structural and functional information for biomembranes and their proteins is by static solid-state NMR of oriented systems. Here, a general procedure to align fully functional biological membranes containing large membrane proteins ( M r >30,000) is described. The method, based on the isopotential spin-dry ultracentrifugation technique, relies on the centrifugation of membrane fragments onto a support with simultaneous, or subsequent, partial evaporation of the solvent which aids alignment. The quality of orientation, as shown by the mosaic spread of the samples, was monitored by static solid-state 31 P NMR for the phospholipids and by 2 H NMR for a deuterated retinal in bovine rhodopsin. The generality of this method is demonstrated with three different membranes containing bovine rhodopsin in reconstituted bilayers, natural membranes with the red cell anion exchange transport protein in erythrocytes, band 3, and the nicotinic acetylcholine receptor.

An efficient NMR methods for the characterisation of 14N sites has been developed with efficiencies suitable for the quantitative analysis of biomolecular and natural abundance systems., Nitrogen is one of the most abundant elements and plays a key role in the chemistry of biological systems. Despite its widespread distribution, the study of the naturally occurring isotope of nitrogen, 14N (99.6%), has been relatively limited as it is a spin-1 nucleus that typically exhibits a large quadrupolar interaction. Accordingly, most studies of nitrogen sites in biomolecules have been performed on samples enriched with 15N, limiting the application of NMR to samples which can be isotopically enriched. This precludes the analysis of naturally occurring samples and results in the loss of the wealth of structural and dynamic information that the quadrupolar interaction can provide. Recently, several experimental approaches have been developed to characterize 14N sites through their interaction with neighboring ‘spy’ nuclei. Here we describe a novel version of these experiments whereby coherence between the 14N site and the spy nucleus is mediated by the application of a moderate rf field to the 14N. The resulting 13C/14N spectra show good sensitivity on natural abundance and labeled materials; whilst the 14N lineshapes permit the quantitative analysis of the quadrupolar interaction.

The membrane of the Golgi apparatus is a complex mixture of lipids and proteins. In the present work we describe multiscale molecular dynamics simulations of transmembrane protein domains in model membranes that represent the in vivo Golgi environment. The transmembrane domain of glycosyltransferases is required for their correct sorting within the Golgi apparatus. The hydrophobic thickness and oligomerization state of the transmembrane domains have been proposed to mediate this sorting. Fukutin, is a putative Golgi glycosyltransferase implicated in muscular dystrophy.We employ atomistic and coarse-grained molecular dynamics simulations to investigate the stability and membrane interactions of the fukutin transmembrane domain, using various models of the membrane. Our atomistic simulations reveal that the fukutin transmembrane domain can exist as a stable α helix irrespective of the headgroup charge, fatty acid saturation or hydrophobic thickness of the lipid bilayer. Coarse-grained simulations reveal that the tilt angle of the fukutin transmembrane domain is highly variable and dependent upon its local environment; both the hydrophobic thickness and the headgroup charge of the lipid bilayer can alter the tilt angle of the protein. Lastly, we study the dynamics of the fukutin transmembrane domain in a mixed lipid bilayer whose composition closely mimics the complex lipid headgroup composition of the Golgi apparatus.

The activity of integral membrane proteins has long been known to be tightly coupled to the lipid composition of the surrounding lipid bilayer. More recently though the presence of non-annular lipid binding sites have been shown to play a key role in the regulation of membrane channels. In particular recent fluorescence studies have revealed that gating of the potassium channel KcsA is highly dependent on the binding of anionic lipids to three or more non-annular lipid binding sites at the lipid protein interface1. Here we present solid-state NMR studies on KcsA reconstituted into charged lipid bilayers composed of POPC/POPG. These studies are allowing us to investigate the nature of the interaction between the surrounding lipid and these binding sites.Employing 1H-31P saturation transfer MAS NMR2 we have been able to probe the proximity and rate of exchange of lipid in close proximity to the KcsA. A significant attenuation of the POPC resonance was observed upon the saturation of amide protons suggesting that POPC populates the annular sites of KcsA and is in relatively fast exchange with the bulk lipid. In contrast no such attenuation was observed for the POPG, which in light of earlier fluorescence studies suggests that the POPG remains resident at the lipid protein interface and does not readily exchange with the bulk lipid. Preliminary heteronuclear correlation spectra in conjunction with T2 filtering are beginning to provide us with insights into the types of residues involved in this interaction.1) P. Marius et al., Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening. Biophysical Journal 2008 (94)1689-98.2) O. Soubias et al., Evidence for lipid specificity in lipid-rhodopsin interactions Journal of Biological Chemistry 2006 (281)33233-41.

Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of alpha-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi. To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be performed. Expressing the N-terminal domain of Fukutin-1 fused to a His(6) tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4 mg per litre of minimal media. Preliminary biophysical analyses have confirmed the identity of the peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured alpha-helix as predicted from the sequence.

The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channel's subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening., Graphical abstract Highlights ► Solid-state 31P MAS NMR studies have been used to characterise binding to the non-annular binding site of KcsA. ► Residues R64 and R89 have been identified as important in the recognition of anionic lipids in the non-annular binding site. ► Single-channel current recordings demonstrate the functional importance of these interactions for KcsA activity.

The interaction between integral membrane proteins and the lipid bilayer is vital in the regulation of many biological processes. Here we report preliminary solid-state NMR studies on two systems where interactions at the lipid/protein interface play an important role in the regulation and trafficking of integral membrane proteins.The anaesthetic octanol has been shown to interact with several sites on the nicotinic acetylcholine receptor (nAChR) one of which is in the lipid/protein interface. In preliminary studies we are investigating how octanol interacts with the lipid bilayer. Analysis of the 1H-13C dipolar couplings present in 2D dipolar/chemical shift spectra we have been able to measure changes in bilayer fluidity at pharmacologically relevant concentrations. We have been able to correlate these results with 2D-1H-NOE-MAS spectra enabling us to locate the position of the octanol within the lipid bilayer. As these methods function with isotopes at natural abundance levels, we are currently applying them to native nAChR membranes to see if changes in the order parameter profile observed in model lipid bilayers are reproduced in native receptor membranes.The interaction of proteins with lateral domains (rafts) in lipid bilayers has been proposed to play an important role in the trafficking of proteins within cells. We are utilizing exchange spectroscopy to study the partitioning of Fukutin, a protein linked to the onset of muscular dystrophy, into lipid rafts. Exchange measurements have enabled us to observe the restricted lateral diffusion of phospholipids in the presence of lateral domains formed in vesicles formed from ternary lipid mixtures. The affects of these lateral domains on the structure and oligomeric state of the Fukutin is also being probed.

Bicelles (bilayered micelles) are popular model membranes used in the study of peptide structure and membrane interactions. They are traditionally made of long- (C14) and short- (C6) chain phospholipids, namely dimyristoylphosphatidylcholine (D14PC) and dihexanoyl-PC (D6PC). They are attractive membrane mimetics because of their composition and planar surface similar to the native membrane environment. To improve the solubilization of membrane proteins (MPs) and allow their study in bicellar systems, D6PC was replaced by detergents from the monoalkylphosphocholine (MAPCHO) family of which dodecylphosphocholine (DPC) is known for its ability to solubilize MPs. More specifically DPC, tetradecyl- (TPC) and hexadecylPC (HPC) have been employed. To verify the possibility of making bicelles with different hydrophobic thickness to better accommodate MPs, D14PC was also replaced by phospholipids with different acyl chain lengths: dilauroylPC (D12PC), dipalmitoylPC (D16PC) and distearoylPC (D18PC). Preliminary results obtained by 31P solid-state NMR at several lipid-to-detergent molar ratios (q) and temperatures indicate that magnetically-oriented bicelles can be formed with D12PC/DPC (q=2, 17-47°C), D14PC/DPC (q=2-3, 32-52°C), D16PC/DPC (q=1.6-2.4, 42-47°C), D14PC/TPC (q=2, 32-57°C) and D16PC/TPC (q=1.6-2.4, 42-52°C). The temperature range at which these bicelles orient is, thus, dictated by the gel-to-fluid phase transition temperature of the phospholipids. Moreover, the longer the phospholipid chain length, the smaller the q ratio range at which bicelles orient. These results will be discussed in terms of PCs and MAPCHOs solubility. These are promising model membranes that could be amenable to both solution- and solid-state NMR, thus enabling structure determination with different bilayer thickness as well as the study of lipid interactions with a single membrane mimicking system.

Fukutin-I is a member of a family of putative O-linked glycosyltransferases involved in the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to number of congenital muscular dystrophies and are characterised by the hypoglycosylation of the alpha-dystroglycan. The function of these proteins relies critically on their location within the endoplasmic reticulum (ER)/Golgi Apparatus (GA). The retention of proteins in these compartments is thought to be mediated by the interaction of the N-terminal transmembrane domain with the atypical lipid composition of these compartments although the molecular basis for this retention is far from clear. To address this question, we are currently studying how the composition of the lipid bilayer within the ER/GA modifies the structure, oligomeric state and lateral segregation of these proteins. Preliminary studies have established an expression system for the production and labelling of the transmembrane domains of these proteins. Using a combination of liquid and solid-state NMR we are characterising how the structure of the N-terminal transmembrane domain of Fukutin-I varies as a function of lipid/detergent composition. Solid-state NMR methods are revealing how the lipid bilayer composition changes the dynamics of the protein within the bilayer thus providing insights into the proteins oligomeric state. In conjunction with cross-linking and fluorescence resonance energy transfer measurements these studies are revealing significant differences in the oligomeric state of the N-terminal transmembrane domain of Fukutin-I in response to bilayer composition with implications for the regulation of protein trafficking.

Hepatitis C is an RNA virus that replicates in association with intracellular membranous structures call membranous webs (MWs). Viral protein NS4B is a key organizer of replication, one crucial function being the induction of MWs. The mechanisms of MW formation are unknown, but it clearly involves induction of membrane curvature, which may require NS4B oligomerisation and possibly hydrophobic wedging. NS4B is known to oligomerise, and the N-terminal amphipathic helix AH2 has been implied as a major determinant of self-association. In order to understand the process of MW induction, we aimed to determine AH2's capacity to remodel membranes by studying the interaction of AH2 with membranes mimicking those found within the cell using 2H and 31P solid-state NMR. Our results show changes in membrane morphology, induced by AH2 in negatively charged vesicles, an effect not observed in neutral bilayers indicating a requirement for negatively charged lipids. Chemical cross-linking studies of AH2 in lipids vesicles confirms AH2 homo-oligomerisation and suggests a charge dependency; with larger oligomers observed in neutral lipid bilayers compared to negatively charged lipid bilayers and lipid mixtures mimicking cellular membranes. These results suggest that AH2 plays a crucial role in NS4B's capacity to alter membrane morphology.

The long QT syndrome (LQTS) is a cardiac dysfunction that prolongs the heart repolarisation interval, leading to cardiac arrhythmia or failure. This pathology can be induced by off-target effects of drugs which block the human ether-a-go-go related gene (hERG) potassium channels located in the myocardium cell membranes. To reduce the risks of this acquired LQTS, regulatory authorities demand in vitro testing of all new drug entities for hERG-blocking potential. As the pore domain of the hERG channel is an important target of LQTS-prone drugs, the objective of our work was to express and purify this region (Asp540-Tyr673) in E. coli to allow biophysical and electrophysiological studies. The detergent sarkosyl was employed for the solubilisation and a His6 N-terminal tag was used to isolate the transmembrane pore domain of hERG with yields of approximately 0.5-1 mg per liter of LB media. Mass spectrometry and Western blot confirmed the identity of the protein and circular dichroism showed that the majority of the hERG pore domain segments adopt an α-helix structure as was expected from sequence homology with other K+ channels. The functionality of the channel is proven by incorporating it into lipid bilayers formed using Montal Mueller method.

The fukutins are a family of eukaryotic membrane proteins whose mis-localisation within the cell has been shown to be associated with the onset of Fukuyama muscular dystrophy. The interaction between the N-terminal transmembrane domain fukutin with the atypical lipid bilayer of the Golgi apparatus has been postulated to play a crucial role in the retention of the protein there.To investigate the nature of these interactions we have developed a bacterial expression system for isolating the N-terminal transmembrane domain of Fukutin-I. We have characterised this hydrophobic peptide using molecular biology techniques, mass spectrometry, circular dichroism and nuclear magnetic resonance studies.Currently, we are using solid state NMR and fluorescence studies to analyse how the oligomeric state, lateral segregation and structure of fukutin reconstituted into artificial bilayers varies as a function of varying lipid compositions. These studies will provide further insight into the role of lipid-protein interactions in membrane protein structure and assembly, segregation into lipid rafts and the role these properties may play in protein trafficking.

Serum amyloid-P component is found ubiquitously in amyloid deposits and has been shown to stabilize fibrillar structures and prevent clearance by the host's defences. Here we report on solid-state NMR studies on fibrillar deposits formed from β2-microglobulin, typically found in patients with dialysis related amyloidosis, and their interactions with serum amyloid-P component.We have successfully undertaken the expression, purification and refolding of the 99 residue β2-microglobulin and established conditions for optimal binding of serum amyloid-P component. High resolution solid-state magic-angle spinning (MAS)-NMR spectra obtained from the fibrils indicate that within the fibrils the β2-microglobulin adopts a homogeneous structure. Using two-dimension homo- and hetero-nuclear correlation spectroscopy we have been able to assign several of the sites within the protein. Currently we are using a range of labelling schemes and acquiring three-dimensional data-sets to complete this assignment. Comparison of the assignment with that obtained from monomeric β2-microglobulin in solution is beginning to provide valuable insights into the structural changes occurring upon fibrilization. Similar comparisons with fibres decorated with serum amyloid-P component should provide valuable insights into how this molecule interacts and stabilizes amyloid fibrils at a molecular level.During the course of these experiments we have also obtained 2D correlation data on inclusion bodies of β2-microglobulin. The resolution attained is not as high as that observed in the fibrillar spectra, however they permit the assignment of resonances to amino acids with the β2-microglobulin. This suggests that within the inclusion bodies the β2-microglobulin adopts a well defined conformation with the lower spectral resolution arising from reduction in dynamics in these highly packed structures. Detailed comparisons of the data with that obtained from the soluble and fibrillar β2-microglobulin should provide insights into the nature of this structure.