Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.     

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and ³¹P, ¹³C, and ¹H nuclear magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5–, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and ¹³C- and ¹H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% -helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of -helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. Correspondence to: G. L. Mendz     

Interaction of myelin basic protein with micelles of dodecylphosphocholine

Interactions of myelin basic protein (MBP) and peptides derived from it with micelles of dodecylphosphocholine (DPC) and perdeuterated DPC have been studied by proton nuclear magnetic resonance (NMR) at 400 MHz and by circular dichroism (CD). When MBP binds to DPC micelles, it acquires about 18% alpha-helicity. The CD spectra of various peptides derived by cleavage of MBP indicate that a major alpha-helical region occurs in residues 85-99 just before the sequence of three prolyl residues 100-102. From line broadenings by fatty acid spin-labels in the micelles and from changes in chemical shifts, the NMR data identify specific residues in MBP that participate in lipid binding. One such sequence is an alpha-helical region from residues 85 to 95, and others occur around methionine-21 and between residues 117 and 135. The different effects of C5, C12, and C16 spin-labels suggest that some segments of the protein may penetrate beyond the dipolar interfacial region of the micelles into the hydrophobic interior, but no part of the protein is protected by the micelles against rapid exchange of its amide groups with the aqueous environment. Even at a lipid to protein molar ratio of 200/1, most NMR resonances from side chains of amino acid residues are not appreciably broadened, suggesting that much of the polypeptide remains highly mobile.     

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.     

Comparing the structural dynamics of the human KCNE3 in reconstituted micelle and lipid bilayered vesicle environments

KCNE3 is a single transmembrane protein of the KCNE family that modulates the function and trafficking of several voltage-gated potassium channels, including KCNQ1. Structural studies of KCNE3 have been previously conducted in a wide range of model membrane mimics. However, it is important to assess the impact of the membrane mimics used on the observed conformation and dynamics. In this study, we have optimized a method for the reconstitution of the KCNE3 into POPC/POPG lipid bilayer vesicles for electron paramagnetic resonance (EPR) spectroscopy. Our CD spectroscopic data suggested that the degree of regular secondary structure for KCNE3 protein reconstituted into lipid bilayered vesicle is significantly higher than in DPC detergent micelles. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) was used to probe the structural dynamics of S49C, M59C, L67C, V85C, and S101C mutations of KCNE3 in both DPC micelles and in POPC/POPG lipid bilayered vesicles. Our CW-EPR power saturation data suggested that the site S74C is buried inside the lipid bilayered membrane while the site V85C is located outside the membrane, in contrast to DPC micelle results. These results suggest that the KCNE3 micelle structures need to be refined using data obtained in the lipid bilayered vesicles in order to ascertain the native structure of KCNE3. This work will provide guidelines for detailed structural studies of KCNE3 in a more native membrane environment and comparing the lipid bilayer results to the isotropic bicelle structure and to the KCNQ1-bound cryo-EM structure.     

Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine: EPR and NMR studies

The spin labels, 5-doxylstearate, 12-doxylstearate, 16-doxylstearate and 1-oxyl-2,2,6,6-tetramethyl-4-dodecylphosphopiperidine, have been incorporated into dodecylphosphocholine micelles and mixed dodecylphosphocholine glucagon micelles. The EPR spectral parameters for the different spin labels and the 1H- and 13C-NMR relaxation rates for nuclei of the detergent molecules indicated that inclusion of up to one spin label molecule per micelle had little influence on the spatial organization of the micelles. Furthermore, the location and environment of the spin labels in the dodecylphosphocholine micelles were not noticeably affected by the addition of glucagon and the 1H-NMR spectra observed for glucagon in mixed spin label/deuterated dodecylphosphocholine/glucagon micelles showed that the different spin labels had essentially no effect on the conformation of glucagon. Approximate spatial locations within the micelle for the nitroxide moieties of the different spin labels were determined from the NMR relaxation rates observed for different nuclei of dodecylphosphocholine. On this basis, the line broadening of individually assigned glucagon 1H-NMR lines by the different spin labels was used to determine the approximate orientation of the polypeptide chain with respect to the micelle surface. Overall, the data indicate that the glucagon backbone runs roughly parallel to the micelle surface, with the depth of immersion adjusted so that polar and apolar side chains can be oriented towards the surface or interior of the micelle, respectively.     

Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine EPR and NMR studies

The spin labels, 5-doxylstearate, 12-doxylstearate, 16-doxylstearate and 1-oxyl-2,2,6,6-tetramethyl-4-dodecylphospiperidine, have been incorporated into dodecylphospocholine micelles and mixed dodecylphosphocholine/ glucagon micelles. The EPR spectral parameters for the different spin labels and the ¹H- and ¹³C-NMR relaxation rates for nuclei of the detergent molecules indicated that inclusion of up to one spin label molecule per micelle had little influence on the spatial organization of the micelles. Furthermore, the location and environment of the spin labels in the dodecylphosphocholine micelles were not noticeably affected by the addition of glucagon and the ¹H-NMR spectra observed for glucagon in mixed spin label/deuterated dodecylphosphocholine/glucagon micelles showed that the different spin labels had essentially no effect on the conformation of glucagon. Approximate spatial locations within the micelle for the nitroxide moieties of the different spin labels were determined from the NMR relaxation rates observed for different nuclei of dodecylphosphocholine. On this basis, the line broadening of individually assigned glucagon ¹H-NMR lines by the different spin labels was used to determine the approximate orientation of the polypeptide chain with respect to the micelle surface. Overall, the data indicate that the glucagon backbone runs roughly parallel to the micelle surface, with the depth of immersion adjusted so that polar and apolar side chains can be oriented towards the surface or interior of the micelle, respectively.     

Spontaneous formation of detergent micelles around the outer membrane protein OmpX

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of approximately 18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic beta-barrel of OmpX rather than in a micellar-like oblate; approximately 40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the beta-barrel region. The phospholipids bind also on the extracellular, protruding beta-sheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, approximately 90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding beta-sheet as a "fishing rod".     

Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID-BA or NSAID-BA-PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA-IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA-IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA-IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA-IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.     

Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

The polar headgroup of the detergent governs the accessibility to water of tryptophan octyl ester in host micelles

Many attempts have been made to rationalize the use of detergents for membrane protein studies [J. Biol. Chem. 264 (1989) 4907]. The barrier properties of the detergent headgroup may be one parameter critically involved in protein protection. In this paper, we analyzed these properties using a model system, by comparing the accessibility of tryptophan octyl ester (TOE) to water-soluble collisional quenchers (iodide and acrylamide) in three detergent micelles. The detergents used differed only in the chemical nature of their polar headgroups, zwitterionic for dodecylphosphocholine (DPC) and nonionic for octa(ethylene glycol) dodecyl monoether (C(12)E(8)) and dodecylmaltoside (DM). In all cases, in phosphate buffer at pH 7.5, the binding of 5 microM TOE was complete in the presence of a slight excess of detergent micelles over TOE molecules, resulting in a significant blue shift and greater intensity of TOE fluorescence emission. The resulting quantum yield of bound TOE was between 0.08 (in DPC) and 0.12 (in DM) with an emission maximum (lambda(max)) of approximately 335 nm whatever the detergent micelle. Time-resolved fluorescence intensity decays of TOE at lambda(max) were heterogeneous in all micelles (3-4 lifetime populations), with mean lifetimes of 1.7 ns in DPC, and 2 ns in both C(12)E(8) and DM. TOE fluorescence quenching by iodide, in detergent micelles, yielded linear Stern-Volmer plots characteristic of a dynamic quenching process. The accessibility of TOE to this ion was the greatest with C(12)E(8), followed by DPC and finally DM (Stern-Volmer quenching constants K(sv) of 2 to 5.5 M(-1)). In contrast, the accessibility of TOE to acrylamide was greatest with DPC, followed by C(12)E(8) and finally DM (K(sv)=2.7-7.1 M(-1)). TOE also presents less rotational mobility in DM than in the other two detergents, as shown from anisotropy decay measurements. These results, together with previous TOE quenching measurements with brominated detergents [Biophys. J. 77 (1999) 3071] provide reference data for analyzing Trp characteristics in peptide (and more indirectly protein)-detergent complexes. The main finding of this study was that TOE was less accessible (to soluble quenchers) in DM than in DPC and C(12)E(8), the cohesion of DM headgroup region being suggested to play a role in the ability of this detergent to protect function and stability of solubilized membrane proteins.     

pH-Dependent conformational changes and topology of a herpesvirus translocating peptide in a membrane-mimetic environment

Pol peptide, an oligopeptide corresponding to the 27 C-terminal amino acids of DNA polymerase from herpes simplex virus type 1, has recently been suggested to translocate from endosomal compartments into the cytosol after being intracellularly delivered via a protein carrier. While an acidic environment was thought to be important for Pol peptide membrane translocation, the mechanism of translocation remains unclear. To investigate the influence of an acidic environment on the conformational properties of the peptide and on its propensity to interact with lipid bilayers, we characterized the structure of Pol peptide at different pH values by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The influence of detergent micelles, which mimic biological lipid membranes, on the peptide secondary structure was also studied. Our CD results indicate that the peptide is in a random conformation in aqueous solution at both acidic and basic pH, whereas in the presence of dodecylphosphocholine (DPC) micelles, it assumes a partial alpha-helical structure which is significantly pH-dependent. An NMR study confirmed that, in the presence of DPC micelles, a short C-terminal alpha-helix is present at pH 6.5, whereas almost two-thirds of the peptide (residues 10-26) fold into an extended amphipathic alpha-helix at pH 4.0. The orientation of Pol peptide relative to the DPC micelle was investigated using paramagnetic probes at both pH 4.0 and 6.5. These studies show that the peptide inserts deeply into the micelle at pH 4.0, whereas it is more exposed to the aqueous environment at pH 6.5. On the basis of these results, a model which might explain the mechanism of translocation of Pol peptide from acidic endosomes to the cytosol is discussed.     

Investigation of the binding geometry of a peripheral membrane protein

A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three-dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of the early endosome antigen 1 (EEA1) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin-labeled probes. The 5- and 14-doxyl-phosphatidylcholine spin-labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (PI3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin-label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the nonpolar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin-label NMR approach for investigating the binding geometry by peripheral membrane proteins.     

The interaction of beta-amyloid protein fragment (12-28) with lipid environments.

The neurotoxicity of beta-amyloid protein (beta AP) fragments may be a result of their solution conformation, which is very sensitive to solution conditions. In this work we describe NMR and CD studies of the conformation of beta AP(12-28) in lipid (micelle) environments as a function of pH and lipid type. The interaction of beta AP(12-28) with zwitterionic dodecylphosphocholine (DPC) micelles is weak and alters the conformation when compared to water solution alone. By contrast, the interaction of the peptide with anionic sodium dodecylsulfate (SDS) micelles is strong: beta AP(12-28) is mostly bound, is alpha-helical from K16 to V24, and aggregates slowly. The pH-dependent conformation changes of beta AP(12-28) in solution occur in the pH range at which the side-chain groups of E22, D23, H13, and H14 are deprotonated (pKas ca. 4 and 6.5); the interaction of beta AP(12-28) with SDS micelles alters the pH-dependent conformational transitions of the peptide whereas the weak interaction with DPC micelles causes little change.     

Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis

A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.     

Structure and dynamics of micelle-associated human immunodeficiency virus gp41 fusion domain

The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.     

Dissociation, aggregation of sesame alpha-globulin in nonionic detergent solution.

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame alpha-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pKInt value of the tyrosyl groups at 1x10-3 M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.     

NMR studies in dodecylphosphocholine of a fragment containing the seventh transmembrane helix of a G-protein-coupled receptor from Saccharomyces cerevisiae

The structure and dynamics of a large segment of Ste2p, the G-protein-coupled alpha-factor receptor from yeast, were studied in dodecylphosphocholine (DPC) micelles using solution NMR spectroscopy. We investigated the 73-residue peptide EL3-TM7-CT40 consisting of the third extracellular loop 3 (EL3), the seventh transmembrane helix (TM7), and 40 residues from the cytosolic C-terminal domain (CT40). The structure reveals the presence of an alpha-helix in the segment encompassing residues 10-30, which is perturbed around the internal Pro-24 residue. Root mean-square deviation values of individually superimposed helical segments 10-20 and 25-30 were 0.91 +/- 0.33 A and 0.76 +/- 0.37 A, respectively. 15N-relaxation and residual dipolar coupling data support a rather stable fold for the TM7 part of EL3-TM7-CT40, whereas the EL3 and CT40 segments are more flexible. Spin-label data indicate that the TM7 helix integrates into DPC micelles but is flexible around the internal Pro-24 site, exposing residues 22-26 to solution and reveal a second site of interaction with the micelle within a region comprising residues 43-58, which forms part of a less well-defined nascent helix. These findings are discussed in light of previous studies in organic-aqueous solvent systems.     

Calcium-modulated S100 protein-phospholipid interactions. An NMR study of calbindin D9k and DPC

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D(9k) (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca(2+), the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca(2+)-loaded calbindin D(9k) does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca(2+) is bound to the protein. A reduction in pH in the absence of Ca(2+) increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca(2+) affinity causes a transition to the micelle-associated state also in the presence of Ca(2+) when the pH is lowered. Site-specific analysis of the data indicates that calbindin D(9k) has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca(2+)-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D(9k) in the cell is discussed, along with the broader implications for the function of the S100 protein family.     

Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation

An understanding of the folding states of α-helical membrane proteins in detergent systems is important for functional and structural studies of these proteins. Here, we present a rapid and simple method for identification of the folding topology and assembly of transmembrane helices using paramagnetic perturbation in nuclear magnetic resonance spectroscopy. By monitoring the perturbation of signals from glycine residues located at specific sites, the folding topology and the assembly of transmembrane helices of membrane proteins were easily identified without time-consuming backbone assignment. This method is validated with Mistic (membrane-integrating sequence for translation of integral membrane protein constructs) of known structure as a reference protein. The folding topologies of two bacterial histidine kinase membrane proteins (SCO3062 and YbdK) were investigated by this method in dodecyl phosphocholine (DPC) micelles. Combing with analytical ultracentrifugation, we identified that the transmembrane domain of YbdK is present as a parallel dimer in DPC micelle. In contrast, the interaction of transmembrane domain of SCO3062 is not maintained in DPC micelle due to disruption of native structure of the periplasmic domain by DPC micelle.