Direct observation and characterization of DMPC/DHPC aggregates under conditions relevant for biological solution NMR

We have used cryo-transmission electron microscopy (cryo-TEM) for inspection of aggregates formed by dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in aqueous solution at total phospholipid concentrations cL < or = 5% and DMPC/DHPC ratios q < or = 4.0. In combination with ocular inspections, we are able to sketch out this part of phase-diagram at T = 14-80 degrees C. The temperature and the ratio q are the dominating variables for changing sample morphology, while cL to a lesser extent affects the aggregate structure. At q = 0.5, small, possibly disc-shaped, aggregates with a diameter of approximately 6 nm are formed. At higher q-values, distorted discoidal micelles that tend to short cylindrical micelles are observed. The more well-shaped discs have a diameter of around 20 nm. Upon increasing q or the temperature, long slightly flattened cylindrical micelles that eventually branch are formed. A holey lamellar phase finally appears upon further elevation of q or temperature. The implications for biological NMR work are two. First, discs prepared as membrane mimics are frequently much smaller than predicted by current "ideal bicelle" models. Second, the q approximately 3 preparations used for aligning water-soluble biomolecules in magnetic fields consist of perforated lamellar sheets. Furthermore, the discovered sequence of morphological transitions may have important implications for the development of bicelle-based membrane protein crystallization methods.     

Bicelles: A natural ‘molecular goniometer’ for structural,dynamical and topological studies of molecules in membranes

Major biological processes occur at the biological membrane. One of the great challenges is to understand the function of chemical or biological molecules inside the membrane; as well of those involved in membrane trafficking. This requires obtaining a complete picture of the in situ structure and dynamics as well as the topology and orientation of these molecules in the membrane lipid bilayer. These led to the creation of several innovative models of biological membranes in order to investigate the structure and dynamics of amphiphilic molecules, as well as integral membrane proteins having single or multiple transmembrane segments. Because the determination of the structure, dynamics and topology of molecules in membranes requires a macroscopic alignment of the system, a new membrane model called ‘bicelles’ that represents a crossover between lipid vesicles and classical micelles has become very popular due to its property of spontaneous self-orientation in magnetic fields. In addition, crucial factors involved in mimicking natural membranes, such as sample hydration, pH and salinity limits, are easy to control in bicelle systems. Bicelles are composed of mixtures of long chain (14–18 carbons) and short chain phospholipids (6–8 carbons) hydrated up to 98% with buffers and may adopt various morphologies depending on lipid composition, temperature and hydration. We have been developing bicelle systems under the form of nano-discs made of lipids with saturated or biphenyl-containing fatty acyl chains. Depending on the lipid nature, these membranous nano-discs may be macroscopically oriented with their normal perpendicular or parallel to the magnetic field, providing a natural ‘molecular goniometer’ for structural and topological studies, especially in the field of NMR. Bicelles can also be spun at the magic angle and lead to the 3D structural determination of molecules in membranes.     

Overexpression,purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies

Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein-protein interactions are as yet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus, recombinant PLB and SLN production in Escherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR

Subunit a in the membrane traversing F₀ sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp ?NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H₂O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.     

Effects of Phe-to-Trp mutation and fluorotryptophan incorporation on the solution structure of cardiac troponin C, and analysis of its suitability as a potential probe for in situ NMR studies

19F NMR spectroscopy is potentially a powerful tool for probing protein properties in situ. However, results obtained using this technique are relevant only if the 19F probe offers minimal perturbation to the surrounding environment. In this paper, we examine the effect of 5-fluorotryptophan (5fW) incorporation on the three-dimensional structure of cardiac troponin-C (cTnC), with the intention of developing a 19F-labeled TnC for use in in situ 19FNMR. We find that, in general, 5fW does not perturb the structure of the protein significantly. Replacement of residue Phe 153 with 5fW produces no noticeable change in protein conformation. However, replacement of residue Phe 104 with 5fW produces a folding behavior that is dependent on the Escherichia coli strain used to express the mutant. The orientations of the indole rings in these mutants are such that the Trp residue adopts a chi2 of approximately 90 degrees in the F104W mutant and approximately -100 degrees in the F153W mutant. Using results from 19F-1H heteronuclear NOE experiment, we show the replacement of L-Trp with 5fW at these positions does not change the orientation of the indole ring and the spread of the 5fW side-chain dihedral angles increases moderately for the F104(5fW) mutant and not at all for the F153(5fW) mutant. Based on these structures, we conclude that the substitution of Phe by 5fW at these two positions has minimal effects on the structure of cTnC and that the 5fW indole rings in both mutants have well defined orientation, making the two mutants viable candidates for use in in situ 19F NMR spectroscopy.     

Use of organic solvents and small molecules for locating binding sites on proteins in solution*

Application of a modified ePHOGSY and other novel NMR experiments to an H2O-DMSO solution of the protein FKBP12 identified the presence of one molecule of DMSO bound in the substrate binding site. It occupies the same spatial region occupied by the pipecolidine moiety of the immunosuppressive drugs FK506 and Rapamycin complexed to the protein. The binding constant KD for this DMSO molecule was only 275 mM. A substructure search of small molecules similar to DMSO resulted in the identification of molecules with improved binding affinity. This work represents a clear example of the powerful interplay of molecular modelling and NMR.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

The effect of amine structure on complexation with lasalocid in model membrane systems. II. Ionophore selectivity for amines in lipid bilayers and at oil/water interfaces

The ionophore antibiotic X-537A (lasalocid) transports biogenic amines across biological and artificial membranes. The major portion of amine flux (greater than 99%) occurs as a 1:1 neutral complex. The rank order of ionophore selectivity was determined for lipid bilayer membrane transport of amines based on a comparison of permeability coefficients: p-tyramine ～ β-phenylethylamine ～ amphetamine > methamphetamine > dopamine > phenylephrine ～ metanephrine > norepinephrine > epinephrine. This rank order is in agreement with results obtained from partitioning measurements which were carried out in parallel to the bilayer membrane experiments. A correlation between amine structure and binding characteristics has been developed.     

Vibrational 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly(alanine-glycine) as model for silk I Bombyx mori fibroin

This study focuses on the conformational characterization of poly(alanine-glycine) II (pAG II) as a model for a Bombyx mori fibroin silk I structure. Raman, IR, and 13C-cross-polarization/magic angle spinning NMR spectra of pAG II are discussed in comparison with those of the crystalline fraction of B. mori silk fibroin (chymotryptic precipitate, Cp) with a silk I (silk I-Cp) structure. The spectral data give evidence that silk I-Cp and the synthetic copolypeptide pAG II have similar conformations. Moreover, the spectral findings reveal that silk I-Cp is more crystalline than pAG II; consequently, the latter contains a larger amount of the random coil conformation. Differential scanning calorimetry measurements confirm this result. N-Deuteration experiments on pAG II allow us to attribute the Raman component at 1320 cm(-1) to the amide III mode of a beta-turn type II conformation, thus confirming the results of those who propose a repeated beta-turn type II structure for silk I. The analysis of the Raman spectra in the nuNH region confirms that the silk I structure is characterized by the presence of different types of H-bonding arrangements, in agreement with the above model.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

The Fluid—Mosaic Model of Membrane Structure: Still relevant to understanding the structure,function and dynamics of biological membranes after more than 40 years

In 1972 the Fluid—Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720–731]. After over 40 years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid–lipid, protein–protein and lipid–protein interactions as well as cell–matrix, cell–cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along with membrane-associated cytoskeletal and extracellular structures maintain the long-range, non-random mosaic macro-organization of membranes, while smaller membrane nano- and submicro-sized domains, such as lipid rafts and protein complexes, are important in maintaining specialized membrane structures that are in cooperative dynamic flux in a crowded membrane plane. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers

Amphipathic helices in membrane proteins that interact with the hydrophobic/hydrophilic interface of the lipid bilayer have been difficult to structurally characterize. Here, the backbone structure and orientation of an amphipathic helix in the full-length M2 protein from influenza A virus has been characterized. The protein has been studied in hydrated DMPC/DMPG lipid bilayers above the gel to liquid-crystalline phase transition temperature by solid-state NMR spectroscopy. Characteristic PISA (Polar Index Slant Angle) wheels reflecting helical wheels have been observed in uniformly aligned bilayer preparations of both uniformly 15N labeled and amino acid specific labeled M2 samples. Hydrogen/deuterium exchange studies have shown the very slow exchange of some residues in the amphipathic helix and more rapid exchange for the transmembrane helix. These latter results clearly suggest the presence of an aqueous pore. A variation in exchange rate about the transmembrane helical axis provides additional support for this claim and suggests that motions occur about the helical axes in this tetramer to expose the entire backbone to the pore.     

StarD4-mediated translocation of 7-hydroperoxycholesterol to isolated mitochondria: Deleterious effects and implications for steroidogenesis under oxidative stress conditions

StAR family proteins, including StarD4, play a key role in steroidogenesis by transporting cholesterol (Ch) into mitochondria for conversion to pregnenolone. Using a model system consisting of peroxidized cholesterol (7α-OOH)-containing liposomes as donors, we showed that human recombinant StarD4 accelerates 7α-OOH transfer to isolated liver mitochondria, and to a greater extent than Ch transfer. StarD4 had no effect on transfer of non-oxidized or peroxidized phosphatidylcholine, consistent with sterol ring specificity. StarD4-accelerated 7α-OOH transfer to mitochondria resulted in greater susceptibility to free radical lipid peroxidation and loss of membrane potential than in a non-StarD4 control. The novel implication of these findings is that in oxidative stress states, inappropriate StAR-mediated trafficking of peroxidized Ch in steroidogenic tissues could result in damage and dysfunction selectively targeted to mitochondria.