Synthesis and use of a pseudo-cysteine for native chemical ligation.

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.     

Synthesis of an O‐acyl isopeptide by using native chemical ligation in an aqueous solvent system

O‐Acyl isopeptides, in which the N‐acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O‐acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O‐acyl isopeptide of 34‐mer pyroGlu‐ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu¹‐ADan(1‐21)‐SCH₂CH₂SO₃^?Na⁺ (3) and Cys²²‐O‐acyl isopeptide (4), in which the amino group of the Ser²⁹ residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O‐acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O‐to‐N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O‐acyl isopeptide, (ii) HPLC purification as the O‐acyl isopeptide form, and (iii) O‐to‐N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Semi‐synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide‐α‐thioester

Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three‐dimensional structure domain was constructed from three segments murine PrP (mPrP)(90–177), mPrP(178–212), and mPrP(213–230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C‐terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

The C1b domain of protein kinase Cδ (PKCδ), a potent receptor for ligands such as diacylglycerol and phorbol esters, was synthesized by utilizing native chemical ligation. With this synthetic strategy, the domain was efficiently constructed and shown to have high affinity ligand binding and correct folding. The C1b domain has been utilized for the development of novel ligands for the control of phosphorylation by PKC family members. This strategy will pave the way for the efficient construction of C1b domains modified with fluorescent dyes, biotin, etc. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of lucifensin by native chemical ligation and characteristics of its isomer having different disulfide bridge pattern

The antimicrobial 40‐amino‐acid‐peptide lucifensin was synthesized by native chemical ligation (NCL) using N‐acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid‐phase synthesis is imcompatible. Two models of ligation were developed: [15 + 25] Ala‐Cys and [19 + 21] His‐Cys. The [19 + 21] His‐Cys method gives lower yield because of the lower stability of 18‐peptide‐His‐Nbz‐CONH₂ peptide, as suggested by density functional theory calculation. Acetamidomethyl‐deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1?6], [C3?5], and [C2?4], which limits its ability to form α‐helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one‐pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Ultrafiltration,a useful method for isolation of intermediates in native chemical ligation exemplified with the total synthesis of Sortase AΔN59

In this paper, ultrafiltration was employed to facilitate the isolation of intermediates in native chemical ligation. Depending on the molecular weight cutoff of the membrane used, molecules with different sizes could be purified, separated, or concentrated by the ultrafiltration process. Total chemical synthesis of the polypeptide chain of the enzyme Sortase A_ΔN59 was used as an example of the application of ultrafiltration in chemical protein synthesis. Sortase A is a ligase that catalyzes transpeptidation reactions between proteins that have C‐terminal LPXTG recognition sequence and Gly5‐ on the peptidoglycan of bacterial cell walls [3]. Ultrafiltration technique facilitated synthesis of Sortase A_ΔN59 and was a promising tool in isolation of intermediates in native chemical ligation. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Total chemical synthesis of dengue 2 virus capsid protein via native chemical ligation: Role of the conserved salt-bridge

The dengue capsid protein C is a highly basic alpha-helical protein of ～100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55′. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C (^WTDEN2C) and its salt bridge-void analog ^E87ADEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

Summary The CC chemokine CCL14/HCC-1(9–74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we, report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9–39) and CCL14/HCC-1(40–74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9–39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

The CC chemokine CCL14/HCC-1(9-74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9-39) and CCL14/HCC-1(40-74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9-39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modelling methods. Part II: Studies in the SDS micelle.

There is much evidence to support the hypothesis that lipids play a role in the interaction of peptide hormones with their membrane receptors. This interaction through change of peptide conformation can facilitate the entry of the hormone into the microenvironment of the receptor. In the present study we have examined the interaction of vasopressin and mesotocin with a lipid-sodium dodecylsulfate (SDS) micelle-using 2D nuclear magnetic resonance (NMR) and theoretical methods. Solution structures of two hormones in solution with SDS were established using the nuclear Overhauser effect (NOE) and the (3)J(NHHalpha) couplings. The amino acid sequences of these peptides are: c[C(1)-Y(2)-F(3)-Q(4)-N(5)-C(6)]-P(7)-R(8)-G(9)-NH(2) ([Arg(8)]vasopressin, AVP) and c[C(1)-Y(2)-I(3)-Q(4)-N(5)-C(6)]-P(7)-I(8)-G(9)-NH(2) (MT). Each of the peptides was found to occur as one stable conformation. AVP adopts the cis configuration on the Cys(1)-Tyr(2) peptide bond, a finding not reported so far. The three-dimensional structures of the two peptides studied were determined by a method that consisted of time-averaged molecular dynamics in an explicit SDS micelle with the parm99 force field in AMBER8.0 package. All calculated structures of the studied peptides form beta-turns in their cyclic parts. The C-terminal fragment of MT is bent, whereas that of AVP is extended.     

Orexin-A is composed of a highly conserved C-terminal and a specific, hydrophilic N-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor.

Orexins-A and B, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX(1)R) and orexin-2 (OX(2)R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and (15)N and (13)C natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III:Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I:Leu16-Ala23 and II:Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX(1)R.     

Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex: A study using charge‐specific chemical modifications

H uman α ‐lactalbumin m ade le thal to t umor cells (HAMLET) and its analogs are partially unfolded protein‐oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge‐specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively‐charged lysine residues to negatively‐charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild‐type α‐lactalbumin‐oleic acid complex. With the addition of OA, the wild‐type and guanidinated α‐lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α‐lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively‐charged basic groups on α‐lactalbumin and the negatively‐charged carboxylate groups on OA molecules play an essential role in the binding of OA to α‐lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Quantitative comparison of the efficiency of antibodies against S1 and S2 subunit of SARS coronavirus spike protein in virus neutralization and blocking of receptor binding: implications for the functional roles of S2 subunit

Neutralizing effects of antibodies targeting the C-terminal stalk (S2) subunit of the spike protein of severe acute respiratory syndrome coronavirus have previously been reported, although its mechanism remained elusive. In this study, high titered mouse antisera against the N-terminal globular (S1) and S2 subunits of the S protein were generated and total immunoglobulin G (IgG) was purified from these antisera. The efficiency of these purified IgGs in virus neutralization and blocking of receptor binding were compared quantitatively using virus neutralization assay and a previously developed cell-based receptor binding assay, respectively. We demonstrated that anti-S1 IgG neutralizes the virus and binds to the membrane associated S protein more efficiently than anti-S2 IgG does. Moreover, both anti-S1 and anti-S2 IgGs were able to abolish the binding between S protein and its cellular receptor(s), although anti-S1 IgG showed a significantly higher blocking efficiency. The unexpected blocking ability of anti-S2 IgG towards the receptor binding implied a possible role of the S2 subunit in virus docking process and argues against the current hypothesis of viral entry. On the other hand, the functional roles of the previously reported neutralizing epitopes within S2 subunit were investigated using an antigen specific antibody depletion assay. Depletion of antibodies against these regions significantly diminished, though not completely abolished, the neutralizing effects of anti-S2 IgG. It suggests the absence of a major neutralizing domain on S2 protein. The possible ways of anti-S2 IgGs to abolish the receptor binding and the factors restricting anti-S2 IgGs to neutralize the virus are discussed.     

Signaling by the human serotonin1A receptor is impaired in cellular model of Smith-Lemli-Opitz Syndrome

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin_1A receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin_1A receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin_1A receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin_1A receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin_1A receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.     

Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin1A receptor: Implications in desmosterolosis

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin_1A receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin_1A receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.