Family resemblances? Ligand binding and activation of family A and B G-protein-coupled receptors

In April 2007, the Biochemical Society held a meeting to compare and contrast ligand binding and activation of Family A and B GPCRs (G-protein-coupled receptors). Being the largest class, Family A GPCRs usually receive the most attention, although a previous Biochemical Society meeting has focused on Family B GPCRs. The aim of the present meeting was to bring researchers of both families together in order to identify commonalities between the two. The present article introduces the proceedings of the meeting, briefly commenting on the focus of each of the following articles. ©The Authors.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Ligand binding and activation of the CGRP receptor

The receptor for CGRP (calcitonin gene-related peptide) is a heterodimer between a GPCR (G-protein-coupled receptor), CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). Models have been produced of RAMP1 and CLR. It is likely that the C-terminus of CGRP interacts with the extracellular N-termini of CLR and RAMP1; the extreme N-terminus of CLR is particularly important and may interact directly with CGRP and also with RAMP1. The N-terminus of CGRP interacts with the TM (transmembrane) portion of the receptor; the second ECL (extracellular loop) is especially important. Receptor activation is likely to involve the relative movements of TMs 3 and 6 to create a G-protein-binding pocket, as in Family A GPCRs. Pro321 in TM6 appears to act as a pivot. At the base of TMs 2 and 3, Arg151, His155 and Glu211 may form a loose equivalent of the Family A DRY (Asp-Arg-Tyr) motif. Although the details of this proposed activation mechanism clearly do not apply to all Family B GPCRs, the broad outlines may be conserved. ©The Authors.

Hydroxyl functionalities of middle rank british coals

Most of the new processes involving the utilisation of coal are based on hydroliquefaction, and in order to assess the suitability of the various coals for this purpose and to characterise coals in general, it is desirable to have a detailed and accurate knowledge of their chemical constitution and reactivity. Also, in the consumption of coals as chemical feed stocks, as in hydroliquefaction, it is advantageous to classify the coals in terms of chemical parameters as opposed to, or in addition to, carbonisation parameters. In view of this it is important to realise the functional groups on the coal hydrocarbon skeleton. In this research it was attempted to characterise coals of various rank (and subsequently their macerals) via methods involving both microwave-driven and bench top derivatisation of the hydroxyl functionalities present in coal. These hydroxyl groups are predominantly in the form of hindered phenolic groups, with other alcoholic groupings being less important, in the coals studied here. Four different techniques were employed, three of which - stannylation, silylation and methylation - were based on in situ analysis. The fourth technique - acetylation - involved derivatisation followed by analysis of a leaving group. The four different techniques were critically compared and it is concluded that silylation is the most promising technique for the evaluation of the hydroxyl content of middle rank coals and coal macerals. Derivatisation via stannylation using TBTO was impeded due to the large steric demand of the reagent and acetylation did not successfully derivatise the more hindered phenolic groups. Three novel methylation techniques were investigated and two of these show great potential. The information obtained from the techniques was correlated together to give a comprehensive insight into the coals and coal macerals studied.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Ligand diffusion on protein surface observed in molecular dynamics simulation

The process of binding of small ligands to dihydrofolate reductase protein has been investigated using all-atom molecular dynamics simulations. The existence of a mechanism that facilitates the search of the binding site by the ligand is demonstrated. The mechanism consists of ligand diffusing on the protein’s surface. It has been discussed in the literature before, but has not been explicitly confirmed for realistic molecular systems. The strength of this nonspecific binding is roughly estimated and found to be essential for the binding kinetics.

Discovering the preferences of physicians with regards to rank-ordered medical documents

The practice of evidence-based medicine involves consulting documents from repositories such as Scopus, PubMed, or the Cochrane Library. The most common approach for presenting retrieved documents is in the form of a list, with the assumption that the higher a document is on a list, the more relevant it is. Despite this list-based presentation, it is seldom studied how physicians perceive the importance of the order of documents presented in a list. This paper describes an empirical study that elicited and modeled physicians' preferences with regard to list-based results. Preferences were analyzed using a GRIP method that relies on pairwise comparisons of selected subsets of possible rank-ordered lists composed of 3 documents. The results allow us to draw conclusions regarding physicians' attitudes towards the importance of having documents ranked correctly on a result list, versus the importance of retrieving relevant but misplaced documents. Our findings should help developers of clinical information retrieval applications when deciding how retrieved documents should be presented and how performance of the application should be assessed. © 2012 Springer-Verlag Berlin Heidelberg.

The Automorphism Group of the Free Algebra of Rank Two

The theorem of Czerniakiewicz and Makar-Limanov, that all the automorphisms of a free algebra of rank two are tame is proved here by showing that the group of these automorphisms is the free product of two groups (amalgamating their intersection), the group of all affine automorphisms and the group of all triangular automorphisms. The method consists in finding a bipolar structure. As a consequence every finite subgroup of automorphisms (in characteristic zero) is shown to be conjugate to a group of linear automorphisms.

Factorizations of some Groups of Lie Type of Lie Rank 4

Еленка Генчева, Цанко Генчев В настоящата работа се разглеждат крайни прости групи G , които могат да се представят като произведение на две свои собствени неабелеви прости подгрупи A и B. Всяко такова представяне G = AB е прието да се нарича факторизация на G, а тъй като множителите A и B са избрани да бъдат прости подгрупи на G, то разглежданите факторизации са известни още като прости факторизации на G. Тук се предполага, че G е проста група от лиев тип и лиев ранг 4 над крайно поле GF (q). Ключови думи: крайни прости групи, групи от лиев тип, факторизации на групи.

Rank and k-nullity of contact manifolds

We prove that the dimension of the 1-nullity distribution N(1) on a closed Sasakian manifold M of rankl is at least equal to 2l−1 provided that M has an isolated closed characteristic. The result is then used to provide some examples of k-contact manifolds which are not Sasakian. On a closed, 2n+1-dimensional Sasakian manifold of positive bisectional curvature, we show that either the dimension of N(1) is less than or equal to n+1 or N(1) is the entire tangent bundle TM. In the latter case, the Sasakian manifold Mis isometric to a quotient of the Euclidean sphere under a finite group of isometries. We also point out some interactions between k-nullity, Weinstein conjecture, and minimal unit vector fields.

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Estudo da associação dos genes RANk, RANKL e OPG com a osteopatia diabética

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