Transmembrane Helical Domain of the Cannabinoid CB1 Receptor

Brain cannabinoid (CB₁) receptors are G-protein coupled receptors and belong to the rhodopsin-like subfamily. A homology model of the inactive state of the CB₁ receptor was constructed using the x-ray structure of β₂-adrenergic receptor (β₂AR) as the template. We used 105 ns duration molecular-dynamics simulations of the CB₁ receptor embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer to gain some insight into the structure and function of the CB₁ receptor. As judged from the root mean-square deviations combined with the detailed structural analyses, the helical bundle of the CB₁ receptor appears to be fully converged in 50 ns of the simulation. The results reveal that the helical bundle structure of the CB₁ receptor maintains a topology quite similar to the x-ray structures of G-protein coupled receptors overall. It is also revealed that the CB₁ receptor is stabilized by the formation of extensive, water-mediated H-bond networks, aromatic stacking interactions, and receptor-lipid interactions within the helical core region. It is likely that these interactions, which are often specific to functional motifs, including the S(N)LAxAD, D(E)RY, CWxP, and NPxxY motifs, are the molecular constraints imposed on the inactive state of the CB₁ receptor. It appears that disruption of these specific interactions is necessary to release the molecular constraints to achieve a conformational change of the receptor suitable for G-protein activation.     

Helical water wires

A helitetrahedral model has been proposed to help explain reports of low-frequency oscillations in pure water following electromagnetic excitation at the hydronium ion cyclotron resonance frequency. The Lorentz force and the intrinsic structure constrain the motion of the H₃O⁺ ion so that it enjoys a unique form of proton-hopping, one whose path is helical. This model may also explain the numerous previously observed cyclotron resonance (ICR) biological couplings for cations other than hydronium by merely substituting hydrogen-bonded versions of these for hydronium in the tetrahedral structure. Thus the effectiveness of resonance stimulation in biological systems is explained in terms of the enhanced conductivity and reduced scattering associated with proton-hopping. It is further shown that the addition of charge-balancing hydroxyl ions act to enable oscillatory electric dipole moments that propagate along the helical axis, giving rise to weak power (≈ femtoWatts) radiation patterns. It is conceivable that the radiation associated with this process may play a role in the interactions at the interface between water and living matter.     

Helical segment analysis of α-helical regions in proteins

The methods suggested earlier for the analysis and representation of protein structural data are now extended to the helical regions in finer details. These enable better handling of characterization of bends and distortions, for which statistical parameters are also developed. Using latest myoglobin data, best experimental parameters for the α-helix are deduced to be r_N = 1.55 (0.13) Å, r = 2.28 (0.12) Å, r_C′ = 1.70 (0.10) Å, r₀ = 2.02 (0.12) Å, ? = 100.5 (2.3)°, and t = 1.495 (0.055) Å.     

Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein

Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.     

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy¹ and two-dimensional (2D) electron crystallography² have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method³, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments⁴, microtubules⁵, amyloid fibers⁶, tobacco mosaic viruses⁷, and bacteria flagella⁸, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid⁹ is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.     

Helical Filaments of Human Dmc1 Protein on Single-Stranded DNA: A Cautionary Tale

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single-stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double-stranded DNA with ∼ 6.4 subunits per turn and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy that the Dmc1 filament formed on single-stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or scanning transmission electron microscopy, to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated.     

Helical twists of collagen model peptides

Average helical twists were calculated by the method of Sugeta and Miyazawa (Biopolymers 1967, 5, 673-679) for all of the collagen model peptides analyzed to date. Calculation of the helical twists of all triplets in each peptide strand provided novel insights for several model peptides. In the (Pro-Pro-Gly)n (n = 9 and 10), the helical twists showed cyclic fluctuations between 40 and 65 degrees with a 20 A period, suggesting that their molecular conformations were close enough to the ideal 7/2-helix to show the helical repeat of 20 A. Rather small helical twists in the guest regions of IBP in complex and T3-785 were attributed to the interaction with Integrin I domain and a relaxed conformation caused by three consecutive triplets lacking imino acid residues, respectively. Although most of the triplets used in this study were imino acid-rich triplets, helical twists were scattered in a wide range from 30 to 70 degrees with an overall average of 52.6 degrees . This distribution of helical twists indicated a strong preference for the 7/2-helical conformation (51.4 degrees ) rather than the 10/3-helical model (36 degrees ).     

Helical crystals of sickle cell hemoglobin

Thin ribbon-like crystals are intermediates in the formation of large crystals of deoxyhemoglobin S from many individual fibers. The thin crystals show foldedover regions when observed by electron microscopy. Some crystals are sufficiently long to have several folds each separated by a distance of about 4.4 μm, suggesting that the crystals are helical in solution. The thickness of the crystals varies from 500 to 900 Å as shown by heavy-metal shadowing and by measurements of the thickness at the crossover point where an edge-on view of the crystal is obtained.     

Use of Helical Wheels to Represent the Structures of Proteins and to Identify Segments with Helical Potential

The three-dimensional structures of alpha-helices can be represented by two-dimensional projections which we call helical wheels. Initially, the wheels were employed as graphical restatements of the known structures determined by Kendrew, Perutz, Watson, and their colleagues at the University of Cambridge and by Phillips and his coworkers at The Royal Institution. The characteristics of the helices, discussed by Perutz et al. (1965), and Blake et al. (1965), can be readily visualized by examination of these wheels. For example, the projections for most helical segments of myoglobin, hemoglobin, and lysozyme have distinctive hydrophobic arcs. Moreover, the hydrophobic residues tend to be clustered in the n +/- 3, n, n +/- 4 positions of adjacent helical turns. Such hydrophobic arcs are not observed when the sequences of nonhelical segments are plotted on the wheels. Since the features of these projections are also distinctive, however, the wheels can be used to divide sequences into segments with either helical or nonhelical potential. The sequences of insulin, cytochrome c, ribonuclease A, chymotrypsinogen A, tobacco mosaic virus protein, and human growth hormone were chosen for application of the wheels for this purpose.     

Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation

ATP-binding cassette transporter (ABC) A1 was increased by apolipoprotein A-I without an increase of its message in THP-1 cells. The pulse label study demonstrated that apoA-I retarded degradation of ABCA1. Similar changes were demonstrated by apoA-II, but the effect of high density lipoprotein was almost negligible on the basis of equivalent protein concentration. Thiol protease inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal (ALLN)) increased ABCA1 and slowed its decay in the cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH(4)Cl showed such effects. The effects of apoA-I and ALLN were additive for the increase of ABCA1, and the apoA-I-mediated cellular lipid release was enhanced by ALLN. The data suggest that ABCA1 is rapidly degraded by a thiol protease(s) in the cells unless helical apolipoproteins in their lipid-free form stabilize ABCA1 by protecting it from protease-mediated degradation.     

Elastic Moduli of Helical Polypeptide Chain Structures

The elastic moduli of the α-helix, polyglycine II, and the parallel-chain and antiparallel-chain pleated sheet structures have been calculated. A Urey-Bradley type of potential was used, extended by the inclusion of hydrogen bond stretching terms where appropriate. In the one instance where a valid comparison with experimental data can be made, viz, α-keratin, the calculations indicate that the matrix component, rather than being amorphous, probably contains an ordered structure of higher modulus than the α-helix.     

Helical tubes of FtsZ from Methanococcus jannaschii

Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric forms in vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein from M. jannaschii to produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(alpha,beta)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ from M. jannaschii that were shown to consist of 'thick'-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++ or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the 'thick'-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of approximately 430 A for Ca++-induced tubes or 590 - 620 A for GMPCPP. However, their assembly in vitro as well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a role in vivo. Their size and stability make them suitable for use in motility assays.     

Helical templating of oligopeptides by cyclodextrin dimers

beta-cyclodextrin-based receptors were synthesized and tested for their ability to induce a helical fold in peptides bearing hydrophobic amino acid residues in the i, i+11- or i, i+14-positions. Circular dichroism experiments revealed that a dimeric beta-cylodextrin receptor synthesized from a [1,1'-biphenyl]-4,4'-dithiol core demonstrated an ability to fold a designed peptide bearing the artificial amino acid L-p-t-butylphenylalanine in the i, i+11-positions, while other dimeric and monomeric receptors failed to do so. Titration studies were performed using both circular dichroism and calorimetry, the analysis of which yielded an apparent K(a) on the order of 10(4)-10(5) M(-1). However, no evidence could be obtained for helical folding with a peptide carrying tryptophan residues in place of the p-t-butylphenylalanine units. Our studies suggest that receptors of this type may be useful in molecular recognition of hydrophobic, already alpha-helical peptides in aqueous solution.