Ellipsometric study of nematic alignment on silicon oxides for display

Vertical Alignment Nematics (VANs) displays are a form of LCDs in which the liquid crystals naturally align vertically to the glass substrates. In spite of their name, the liquid crystal (LC) director is never exactly vertical, rather it always show a small angle with the normal to the sample plane called tilt that may vary throughout the cell bulk. Its values are ultimately determined by the pretilt, defined as the tilt angle on the surfaces in the absence of voltage.

Design of equalized ROADMs devices with flexible bandwidth based on LCoS technology

This paper describes the theory, design, applications and performance of a new Reconfigurable Add-drop Multiplexer (ROADM) with flexible bandwidth allocation. The device can address several wavelengths at the input to four output fibers, according to the holograms stored in a SLM (Spatial Light Modulator), where all the outputs are equalized in power. All combinations of the input wavelengths are possible at the different output fibers. Each fiber has assigned all the signals with the same bandwidth; the possible bandwidths are 12.5GHz, 25GHz, 50GHz and 100GHz, according to ITU-T 694.1 Recommendation. It is possible to route several signals with different bandwidth in real time thanks to Liquid Crystal over Silicon (LCoS) technology.

Silicon oxides as alignment surfaces for vertically-aligned nematics in photonic devices

A comparative study on alignment performance and microstructure of inorganic layers used for liquid crystal cell conditioning has been carried out. The study has focused on two specific materials, SiOx and SiO2, deposited under different conditions. The purpose was to establish a relationship between layer microstructure and liquid crystal alignment. The surface morphology has been studied by FESEM and AFM. An analysis on liquid crystal alignment, pretilt angle, response time, contrast ratio and the conditions to develop backflow effect (significant rise time increase due to pure homeotropic alignment) on vertically-aligned nematic cells has been carried out. A technique to overcome the presence of backflow has been identified. The full comparative study of SiOx and SiO2 layer properties and their influence over liquid crystal alignment and electrooptic response is presented.

Structural requirements for 5-HT2A and 5-HT1A serotonin receptor potentiation by the biologically active lipid oleamide

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and that has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT1A receptor response is disclosed, and a study of the structural features of oleamide required for potentiation of the 5-HT2A and 5-HT1A response to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT2A receptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the Δ9-cis double bond is required, and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well defined relationship requiring small amide substituents, suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity. A less extensive study of the 5-HT1A potentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibit rather than potentiate the 5-HT2A, but not the 5-HT1A, receptor response, further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.

Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower kcat and higher Km for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.

A novel glutamine–RNA interaction identified by screening libraries in mammalian cells

The arginine-rich motif provides a versatile framework for RNA recognition in which few amino acids other than arginine are needed to mediate specific binding. Using a mammalian screening system based on transcriptional activation by HIV Tat, we identified novel arginine-rich peptides from combinatorial libraries that bind tightly to the Rev response element of HIV. Remarkably, a single glutamine, but not asparagine, within a stretch of polyarginine can mediate high-affinity binding. These results, together with the structure of a Rev peptide-Rev response element complex, suggest that the carboxamide groups of glutamine or asparagine are well-suited to hydrogen bond to G-A base pairs and begin to establish an RNA recognition code for the arginine-rich motif. The screening approach may provide a relatively general method for screening expression libraries in mammalian cells.

The role of enzyme distortion in the single displacement mechanism of family 19 chitinases

By using molecular dynamics simulations, we have examined the binding of a hexaNAG substrate and two potential hydrolysis intermediates (an oxazoline ion and an oxocarbenium ion) to a family 19 barley chitinase. We find the hexaNAG substrate binds with all sugars in a chair conformation, unlike the family 18 chitinase which causes substrate distortion. Glu 67 is in a position to protonate the anomeric oxygen linking sugar residues D and E whereas Asn 199 serves to hydrogen bond with the C2′ N-acetyl group of sugar D, thus preventing the formation of an oxazoline ion intermediate. In addition, Glu 89 is part of a flexible loop region allowing a conformational change to occur within the active site to bring the oxocarbenium ion intermediate and Glu 89 closer by 4–5 Å. A hydrolysis product with inversion of the anomeric configuration occurs because of nucleophilic attack by a water molecule that is coordinated by Glu 89 and Ser 120. Issues important for the design of inhibitors specific to family 19 chitinases over family 18 chitinases also are discussed.

Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

Structural basis of an embryonically lethal single Ala → Thr mutation in the vnd/NK-2 homeodomain

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to −5°C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25°C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix–turn–helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase

To be effective as antiviral agent, AZT (3′-azido-3′-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-Å x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3′OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the β- and γ-phosphate, which plays an important part in phosphate transfer.

A physical basis for protein secondary structure

A physical theory of protein secondary structure is proposed and tested by performing exceedingly simple Monte Carlo simulations. In essence, secondary structure propensities are predominantly a consequence of two competing local effects, one favoring hydrogen bond formation in helices and turns, the other opposing the attendant reduction in sidechain conformational entropy on helix and turn formation. These sequence specific biases are densely dispersed throughout the unfolded polypeptide chain, where they serve to preorganize the folding process and largely, but imperfectly, anticipate the native secondary structure.

A streptavidin mutant with altered ligand-binding specificity

The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 × 106 M−1, two orders of magnitude higher than that for biotin (1 × 104 M−1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 × 104 M−1) was lower than predicted (2.9 × 105 M−1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.