Method to assess packing quality of transmembrane alpha-helices in proteins. 2. Validation by "correct vs misleading" test

We describe a set of tests designed to check the ability of the new "membrane score" method (see the first paper of this series) to assess the packing quality of transmembrane (TM) alpha-helical domains in proteins. The following issues were addressed: (1) Whether there is a relation between the score (S(mem)) of a model and its closeness to the "nativelike" conformation? (2) Is it possible to recognize a correct model among misfolded and erroneous ones? (3) To what extent the score of a homology-built model is sensitive to errors in sequence alignment? To answer the first question, two test cases were considered: (i) Several models of bovine aquaporin-1 (target protein) were built on the structural templates provided by its homologs with known X-ray structure. (ii) Side chains in the spatial models of visual rhodopsin and cytochrome c oxidase were rebuilt based on the backbone scaffolds taken from their crystal structures, and the resulting models were iteratively fitted into the full-atom X-ray conformations. It was shown that the higher the S(mem) value of a model is, the lower its root-mean-square deviation is from the "correct" (crystal) structure of a target. Furthermore, the "membrane score" method successfully identifies the rhodopsin crystal structure in an ensemble of "rotamer-type" decoys, thus providing the way to optimize mutual orientations of alpha-helices in models of TM domains. Finally, being applied to a set of homology models of rhodopsin built on its crystal structure with systematically shifted alignment, the approach demonstrates a prominent ability to detect alignment errors. We therefore assume that the "membrane score" method will be helpful in optimization of in silico models of TM domains in proteins, especially those in GPCRs.     

Elusive structure of HCl monohydrate

The study addresses the structure of crystalline HCl monohydrate which is composed of H3O+ and Cl-. The published x-ray diffraction patterns indicate an element of disorder, the nature of which is debated in the literature and is addressed in the present study. The computational investigations include searches for alternative crystal structures employing an empirical potential, and on-the-fly simulations as implemented in the density functional code QUICKSTEP employing Gaussian basis sets. The experimental work focuses on Fourier-transform infrared (FTIR) spectra of crystal nanoparticles. Simulations of FTIR spectra and of the x-ray diffraction patterns are consistent with crystal monohydrate structure composed of ferroelectric domains, joined by "boundary tissue" of antiferroelectric structure.     

The structural modeling of EF-hand motifs in parvalbumin

Parvalbumin (Parv) is a typical protein with EF-hand motifs that play an important role in many physiological processes. We present a novel free energy to model the skeletal C\(_\alpha \) chain of the protein from the basic principle of mathematics and physics. Starting from the crystal structure of Parv (PDB code 2PVB), we first analyze the profile of the C\(_\alpha \) bond and torsion angles over the segment that contains the secondary structures. Then the parameters in the energy function are evaluated for the helix ABCD fragment that contains two EF-hand domains in Parv. Meanwhile an eight-soliton configuration at the energy minimum is constructed to model the conformation of ABCD fragment. The deviation of the conformation constructed from the model away from the crystal structure is as small as 1.28 Å. The structural modeling stems from the physical energy, which is a benefit relative to the statistics-based or knowledge-based technologies.     

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.     

氰乙基纤维素/二甲基乙酰胺液晶溶液的研究

本文利用Abbe折射仪,热台偏光显微镜,小角光散射等方法研究了氰乙基纤维素/二甲基乙酰胺液晶溶液的形成,形态结构和某些性质。溶液随浓度的增加,从各向同性态经过两相共存状态,转变成为单一的液晶态。该液晶是胆甾型的。溶液的双折射Δn随浓度的增加或温度的降低而增大。在无外力作用时,液晶相由许多无规分布的取向微区组成。液晶溶液受切应力后形成“条纹结构”,大分子链沿切应力方向取向,并在各条纹中排列有序。     

Method to assess packing quality of transmembrane alpha-helices in proteins. 1. Parametrization using structural data

Integral membrane proteins (MPs) are pharmaceutical targets of exceptional importance. Modern methods of three-dimensional protein structure determination often fail to supply the fast growing field of structure-based drug design with the requested MPs' structures. That is why computational modeling techniques gain a special importance for these objects. Among the principal difficulties limiting application of these methods is the low quality of the MPs' models built in silico. In this series of two papers we present a computational approach to the assessment of the packing "quality" of transmembrane (TM) alpha-helical domains in proteins. The method is based on the concept of protein environment classes, whereby each amino acid residue is described in terms of its environment polarity and accessibility to the membrane. In the first paper we analyze a nonredundant set of 26 TM alpha-helical domains and compute the residues' propensities to five predefined classes of membrane-protein environments. Here we evaluate the proposed approach only by various test sets, cross-validation protocols and ability of the method to delimit the crystal structure of visual rhodopsin, and a number of its erroneous theoretical models. More advanced validation of the method is given in the second article of this series. We assume that the developed "membrane score" method will be helpful in optimizing computer models of TM domains of MPs, especially G-protein coupled receptors.     

Investigation of an Unusual Crystal Habit of Hydrochlorothiazide Reveals Large Polar Enantiopure Domains and a Possible Crystal Nucleation Mechanism

The observation of an unusual crystal habit in the common diuretic drug hydrochlorothiazide (HCT), and identification of its subtle conformational chirality, has stimulated a detailed investigation of its crystalline forms. Enantiomeric conformers of HCT resolve into an unusual structure of conjoined enantiomorphic twin crystals comprising enantiopure domains of opposite chirality. The purity of the domains and the chiral molecular conformation are confirmed by spatially revolved synchrotron micro‐XRD experiments and neutron diffraction, respectively. Macroscopic inversion twin symmetry observed between the crystal wings suggests a pseudoracemic structure that is not a solid solution or a layered crystal structure, but an unusual structural variant of conglomerates and racemic twins. Computed interaction energies for molecular pairs in the racemic and enantiopure polymorphs of HCT, and the observation of large opposing unit‐cell dipole moments for the enantiopure domains in these twin crystals, suggest a plausible crystal nucleation mechanism for this unusual crystal habit.     

Influence of lipids on the interfacial disposition of respiratory syncytical virus matrix protein

The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers. This has enabled factors that influence the disposition of the protein with respect to the lipid interface to be characterized. In the absence of sphingomyelin, respiratory syncytial virus matrix protein penetrates monolayers composed of mixtures of phosphocholines with phosphoethanolamines or cholesterol at the air-water interface. In ternary mixtures composed of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol, the protein exhibits two separate behaviors: (1) peripheral association with the surface of sphingomyelin-rich domains and (2) penetration of sphingomyelin-poor domains. Prolonged incubation of the protein with mixtures of phosphocholines and phosphoethanolamines leads to the formation of helical protein assemblies of uniform diameter that demonstrate an inherent propensity of the protein to assemble into a filamentous form.     

Orientational ordering of crystal domains in ionic liquid based mixtures

By in situ observations using simultaneous X-ray diffraction and the DSC (differential scanning calorimetry) method, the effect of water, methanol, ethanol, and benzene on the crystallization has been observed in an ionic liquid (IL)-rich phase. The IL is a hydrophilic ionic liquid, N, N-diethyl-N-methyl-N-2-methoxyethyl ammonium tetrafluoroborate, [DEME][BF4]. At a small amount of the above additional molecules in the IL, the conventional preferred orientation on the Debye rings was seen by the X-ray diffraction. At 0.9 mol % H2O, twinlike crystal domains develop extraordinary on the microdomains. By the "crystal-growth enhancement effect" at a slight amount of water, a composite domain structure, which consists of the large domain and the weakly orientated microdomains, is formed without internal strains. Above 2.9 mol % H2O, the domain structure, however, disappears completely. It is remarkable that, in a thermal cycling experiment using pure [DEME][BF4], the similar composite domain structure appeared. This is also caused by an uptake of a slight amount of water.     

Structural and spectroscopic studies of peptoid oligomers with alpha-chiral aliphatic side chains

Substantial progress has been made in the synthesis and characterization of various oligomeric molecules capable of autonomous folding to well-defined, repetitive secondary structures. It is now possible to investigate sequence-structure relationships and the driving forces for folding in these systems. Here, we present detailed analysis by X-ray crystallography, NMR, and circular dichroism (CD) of the helical structures formed by N-substituted glycine (or "peptoid") oligomers with alpha-chiral, aliphatic side chains. The X-ray crystal structure of a N-(1-cyclohexylethyl)glycine pentamer, the first reported for any peptoid, shows a helix with cis-amide bonds, approximately 3 residues per turn, and a pitch of approximately 6.7 A. The backbone dihedral angles of this pentamer are similar to those of a polyproline type I peptide helix, in agreement with prior modeling predictions. This crystal structure likely represents the major solution conformers, since the CD spectra of analogous peptoid hexamers, dodecamers, and pentadecamers, composed entirely of either (S)-N-(1-cyclohexylethyl)glycine or (S)-N-(sec-butyl)glycine monomers, also have features similar to those of the polyproline type I helix. Furthermore, this crystal structure is similar to a solution NMR structure previously described for a peptoid pentamer comprised of chiral, aromatic side chains, which suggests that peptoids containing either aromatic or aliphatic alpha-chiral side chains adopt fundamentally similar helical structures in solution, despite distinct CD spectra. The elucidation of detailed structural information for peptoid helices with alpha-chiral aliphatic side chains will facilitate the mimicry of biomolecules, such as transmembrane protein domains, in a distinctly stable form.     

Structural Adaptation of a Protein to Increased Metal Stress: NMR Structure of a Marine Snail Metallothionein with an Additional Domain

In this study, we present an NMR structure of the metallothionein (MT) from the snail Littorina littorea (LlMT) in complex with Cd²⁺. LlMT is capable of binding 9 Zn²⁺ or 9 Cd²⁺ ions. Sequence alignments with other snail MTs revealed that the protein is likely composed of three domains. The study revealed that the protein is divided into three individual domains, each of which folds into a single well‐defined three‐metal cluster. The central α2 and C‐terminal β domains are positioned with a unique relative orientation. Two variants with longer and shorter linkers were investigated, which revealed that specific interdomain contacts only occurred with the wild‐type linker. Moreover, a domain‐swap mutant in which the highly similar α1 and α2 domains were exchanged was structurally almost identical. It is suggested that the expression of a three‐domain MT confers an evolutionary advantage on Littorina littorea in terms of coping with Cd²⁺ stress and adverse environmental conditions.     

Structural basis for oxygen sensing and signal transduction of the heme-based sensor protein Aer2 from Pseudomonas aeruginosa

The crystal structure of a truncated Aer2, a signal transducer protein from Pseudomonas aeruginosa, consisting of the heme-containing PAS and di-HAMP domains revealed that a distal tryptophan residue (Trp283) plays an important role in stabilizing the heme-bound O(2) and intra-molecular signal transduction upon O(2) binding.     

Inward-growing self-assembly of colloidal crystal films on horizontal substrates

Colloidal crystal films have been fabricated on solid substrates with a horizontal deposition method. Scanning electron microscope images showed that the colloidal crystal films exhibit ordered face-centered cubic structures in large domains. Optical measurements demonstrated the presence of photonic band gap along the crystallographic [111] direction. The fabrication method described in this paper allows one to rapidly fabricate colloidal crystal films of different thicknesses, which can be controlled by varying colloidal suspension concentration or volume. In addition, the method also works well for growing colloidal crystal films on a hydrophilic solid substrate with a rough surface. Furthermore, the fabrication of colloidal crystal heterostructures has been demonstrated. An inward-growing mechanism responsible for self-assembly of colloidal spheres on horizontal substrates has been proposed to interpret the observed experimental results.     

Two-Dimensional Boronate Ester Covalent Organic Framework Thin Films with Large Single Crystalline Domains for a Neuromorphic Memory Device

Despite the recent progress in the synthesis of crystalline boronate ester covalent organic frameworks (BECOFs) in powder and thin-film through solvothermal method and on-solid-surface synthesis, respectively, their applications in electronics, remain less explored due to the challenges in thin-film processability and device integration associated with the control of film thickness, layer orientation, stability and crystallinity. Moreover, although the crystalline domain sizes of the powder samples can reach micrometer scale (up to ≈1.5 μm), the reported thin-film samples have so far rather small crystalline domains up to 100 nm. Here we demonstrate a general and efficient synthesis of crystalline two-dimensional (2D) BECOF films composed of porphyrin macrocycles and phenyl or naphthyl linkers (named as 2D BECOF-PP or 2D BECOF-PN ) by employing a surfactant-monolayer-assisted interfacial synthesis (SMAIS) on the water surface. The achieved 2D BECOF-PP is featured as free-standing thin film with large single-crystalline domains up to ≈60 μm² and tunable thickness from 6 to 16 nm. A hybrid memory device composed of 2D BECOF-PP film on silicon nanowire-based field-effect transistor is demonstrated as a bio-inspired system to mimic neuronal synapses, displaying a learning–erasing–forgetting memory process.     

Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes

We present a novel platform for investigating the composition-specific interactions of proteins (or other biologically relevant molecules) with model membranes composed of compositionally distinct domains. We focus on the interaction between a mitochondrial-specific lipid, cardiolipin (CL), and a peripheral membrane protein, cytochrome c (cyt c). We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and the lipid of interest, CL, preferentially localizes into one of the domains (the liquid disordered (L(d)) phase). The presence of CL-rich and CL-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to CL-rich domains and observe fascinating morphological transitions within these regions of membrane. CL-rich domains start to form small buds and eventually fold up into a collapsed state. We also observe that cyt c can induce a strong attraction between the CL-rich domains of adjacent vesicles as demonstrated by the development of large osculating regions between these domains. Qualitatively similar behavior is observed when other polycationic proteins or polymers of a similar size and net charge are used instead of cyt c. We argue that these striking phenomena can be simply understood by consideration of colloidal forces between the protein and the membrane. We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria.     

Giant phospholipid/block copolymer hybrid vesicles: mixing behavior and domain formation

Lipids and block copolymers can be individually assembled into unsupported, spherical membranes (liposomes or polymersomes), each having their own particular benefits and limitations. Here we demonstrate the preparation of microscale, hybrid "lipopolymersomes" composed of the common lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and the commercially available copolymer PBd-b-PEO (polybutadiene-b-poly(ethylene oxide)) with the goal of incorporating the advantageous qualities of the unitary systems into mixed-membrane capsules. We investigate the lipopolymersomes using confocal fluorescence microscopy and demonstrate that these hybrid membranes are well mixed on nanoscopic length scales within the permittable compositional windows for hybrid vesicle formation. We measure the intramembrane dynamics and mechanical properties of these hybrid membranes by fluorescence recovery after photobleaching (FRAP) and micropipet aspiration, respectively. For the first time, we demonstrate the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle membrane. This is achieved by the biotinylation of one of the constituent species and cross linking with the protein NeutrAvidin. The resultant domain patterning is dependent upon which component carries the biotin functionality: cross linking of the copolymer species results in domains that ripen into a single, large, copolymer-rich island, and cross linking of the lipids yields many small, "spot-like", lipid-rich domains within a copolymer-rich matrix. We discuss these morphological differences in terms of the fluidity and mechanical properties of the membrane phases and the possible resultant interdomain interactions within the membrane. These heterogeneous hybrid lipopolymersomes could find applications in fields such as targeted delivery, controlled release, and environmental detection assays where these capsules possess the characteristics of biocompatible lipid membranes combined with enhanced mechanical strength and stability from the copolymer matrix.     

The electro-optics of nematic liquid crystals stabilized by a polymer network

Samples of nematics stabilized by a polymer network, which are new composite materials, were prepared. A ZhK-1277 nematic composite and a bisphenyl-A-dimethacrylate monomer were used. Polymerization was conducted via UV radiation. The electro-optic properties, i.e., the dependence of transmittance and the turn-on and turn-off times on the electric voltage and layer thickness, of the resulting material and a pure nematic were studied. The experimental results are explained by the domain structure of the nematic in a polymer network, according to which the liquid crystal in an electro-optic cell is composed of oriented domains separated by thin partitions of the polymer. The size of the domain regions of the liquid crystal is 2 μm.     

Multidomain protein solves the folding problem by multifunnel combined landscape: theoretical investigation of a y-family DNA polymerase

Approximately three-fourths of eukaryotic proteins are composed of multiple independently folded domains. However, much of our understanding is based on single domain proteins or isolated domains whose studies directly lead to well-known energy landscape theory in which proteins fold by navigating through a funneled energy landscape toward native structure ensembles. The degrees of freedom for proteins with multiple domains are many orders of magnitude larger than that for single domain proteins. Now, the question arises: How do the multidomain proteins solve the "protein folding problem"? Here, we specifically address this issue by exploring the structure folding relationship of Sulfolobus solfataricus DNA polymerase IV (DPO4), a prototype Y-family DNA polymerase which contains a polymerase core consisting of a palm (P domain), a finger (F domain), and a thumb domain (T domain) in addition to a little finger domain (LF domain). The theoretical results are in good agreement with the experimental data and lead to several theoretical predictions. Finally, we propose that for rapid folding into well-defined conformations which carry out the biological functions, four-domain DPO4 employs a divide-and-conquer strategy, that is, combining multiple individual folding funnels into a single funnel (domains fold independently and then coalesce). In this way, the degrees of freedom for multidomain proteins are polynomial rather than exponential, and the conformational search process can be reduced effectively from a large to a smaller time scale.     

Structure Elucidation of Helical Aromatic Foldamer–Protein Complexes with Large Contact Surface Areas

The development of large synthetic ligands could be useful to target the sizeable surface areas involved in protein–protein interactions. Herein, we present long helical aromatic oligoamide foldamers bearing proteinogenic side chains that cover up to 450 Ų of the human carbonic anhydrase II (HCA) surface. The foldamers are composed of aminoquinolinecarboxylic acids bearing proteinogenic side chains and of more flexible aminomethyl-pyridinecarboxylic acids that enhance helix handedness dynamics. Crystal structures of HCA-foldamer complexes were obtained with a 9- and a 14-mer both showing extensive protein–foldamer hydrophobic contacts. In addition, foldamer–foldamer interactions seem to be prevalent in the crystal packing, leading to the peculiar formation of an HCA superhelix wound around a rod of stacked foldamers. Solution studies confirm the positioning of the foldamer at the protein surface as well as a dimerization of the complexes.