Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

Responsive hydrophobically associating polymers:a review of structure and properties

Responsive hydrophobically associating polymers can in many ways be considered to be analogous to proteins in their ability to form compact molecules with a defined secondary structure, and hence, functionality. These molecules are characterized by the presence of alternating charged and hydrophobic groups. The balance between charge repulsion and hydrophobic interactions is sensitive to environmental pH and therefore changes in pH produce controllable conformational changes. The change from a charged extended chain to a collapsed uncharged coil structure is sometimes referred to as hypercoiling behaviour and enables the polymer to act as a simple switch between an 'on' and 'off' state. The purpose of this review is to illustrate the structure and behaviour of polymers that exhibit hypercoiling behaviour and to highlight their potential pharmaceutical applications, which in terms of drug delivery is likely to be related to their surface behaviour and solubilizing activity. © 2001 Elsevier Science B.V. All rights reserved.

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Hypercoiling and hydrophobically associating polymers : interfacial synthesis, surface properties and pharmaceutical applications

The objective of the work described was to identify and synthesize a range of biodegradable hypercoiling or hydrophobically associating polymers to mimic natural apoproteins, such as those found in lung surfactant or plasma apolipoproteins. Stirred interfacial polymerization was used to synthesize potentially biodegradable aromatic polyamides (Mw of 12,000-26,000) based on L-Iysine, L-Iysine ethyl ester, L-ornithine and DL-diaminopropionic acid, by reaction with isophthaloyl chloride. A similar technique was used to synthesize aliphatic polyamides based on L-Iysine ethyl ester and either adipoyl chloride or glutaryl chloride resulting in the synthesis of poly(lysine ethyl ester adipamide) [PLETESA] or poly(lysine ethyl ester glutaramide) (Mw of 126,000 and 26,000, respectively). PLETESA was found to be soluble in both polar and non-polar solvents and the hydrophobic/hydrophilic balance could be modified by partial saponification (66-75%) of the ethyl ester side chains. Surface or interfacial tension/pH profiles were used to assess the conformation of both the poly(isophthalamides) and partially saponified PLETESA in aqueous solution. The results demonstrated that a loss of charge from the polymer was accompanied by an initial fall in surface activity, followed by a rise in activity, and ultimately, by polymer precipitation. These observations were explained by a collapse of the polymer chains into non-surface active intramolecular coils, followed by a transition to an amphipathic conformation, and finally to a collapsed hydrophobe. 2-Dimensional NMR analysis of polymer conformation in polar and non-polar solvents revealed intramolecular associations between the hydrophobic groups within partially saponified PLETESA. Unsaponified PLETESA appeared to form a coiled structure in polar solvents where the ethyl ester side chains were contained within the polymer coil. The implications of the secondary structure of PLETESA and potential biomedical applications are discussed.

Phenotypic effect of mutations in evolving populations of RNA molecules

Background. The secondary structure of folded RNA sequences is a good model to map phenotype onto genotype, as represented by the RNA sequence. Computational studies of the evolution of ensembles of RNA molecules towards target secondary structures yield valuable clues to the mechanisms behind adaptation of complex populations. The relationship between the space of sequences and structures, the organization of RNA ensembles at mutation-selection equilibrium, the time of adaptation as a function of the population parameters, the presence of collective effects in quasispecies, or the optimal mutation rates to promote adaptation all are issues that can be explored within this framework. Results. We investigate the effect of microscopic mutations on the phenotype of RNA molecules during their in silico evolution and adaptation. We calculate the distribution of the effects of mutations on fitness, the relative fractions of beneficial and deleterious mutations and the corresponding selection coefficients for populations evolving under different mutation rates. Three different situations are explored: the mutation-selection equilibrium (optimized population) in three different fitness landscapes, the dynamics during adaptation towards a goal structure (adapting population), and the behavior under periodic population bottlenecks (perturbed population). Conclusions. The ratio between the number of beneficial and deleterious mutations experienced by a population of RNA sequences increases with the value of the mutation rate µ at which evolution proceeds. In contrast, the selective value of mutations remains almost constant, independent of µ, indicating that adaptation occurs through an increase in the amount of beneficial mutations, with little variations in the average effect they have on fitness. Statistical analyses of the distribution of fitness effects reveal that small effects, either beneficial or deleterious, are well described by a Pareto distribution. These results are robust under changes in the fitness landscape, remarkably when, in addition to selecting a target secondary structure, specific subsequences or low-energy folds are required. A population perturbed by bottlenecks behaves similarly to an adapting population, struggling to return to the optimized state. Whether it can survive in the long run or whether it goes extinct depends critically on the length of the time interval between bottlenecks. © 2010 Stich et al; licensee BioMed Central Ltd.

G-protein-coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

Effects of pixel noise and stimulus structure on visual detection performance

This thesis consisted of two major parts, one determining the masking characteristics of pixel noise and the other investigating the properties of the detection filter employed by the visual system. The theoretical cut-off frequency of white pixel noise can be defined from the size of the noise pixel. The empirical cut-off frequency, i.e. the largest size of noise pixels that mimics the effect of white noise in detection, was determined by measuring contrast energy thresholds for grating stimuli in the presence of spatial noise consisting of noise pixels of various sizes and shapes. The critical i.e. minimum number of noise pixels per grating cycle needed to mimic the effect of white noise in detection was found to decrease with the bandwidth of the stimulus. The shape of the noise pixels did not have any effect on the whiteness of pixel noise as long as there was at least the minimum number of noise pixels in all spatial dimensions. Furthermore, the masking power of white pixel noise is best described when the spectral density is calculated by taking into account all the dimensions of noise pixels, i.e. width, height, and duration, even when there is random luminance only in one of these dimensions. The properties of the detection mechanism employed by the visual system were studied by measuring contrast energy thresholds for complex spatial patterns as a function of area in the presence of white pixel noise. Human detection efficiency was obtained by comparing human performance with an ideal detector. The stimuli consisted of band-pass filtered symbols, uniform and patched gratings, and point stimuli with randomised phase spectra. In agreement with the existing literature, the detection performance was found to decline with the increasing amount of detail and contour in the stimulus. A measure of image complexity was developed and successfully applied to the data. The accuracy of the detection mechanism seems to depend on the spatial structure of the stimulus and the spatial spread of contrast energy.

Detection of signals corrupted by the combination of Gaussian and Non-Gaussian noise

The detection of signals in the presence of noise is one of the most basic and important problems encountered by communication engineers. Although the literature abounds with analyses of communications in Gaussian noise, relatively little work has appeared dealing with communications in non-Gaussian noise. In this thesis several digital communication systems disturbed by non-Gaussian noise are analysed. The thesis is divided into two main parts. In the first part, a filtered-Poisson impulse noise model is utilized to calulate error probability characteristics of a linear receiver operating in additive impulsive noise. Firstly the effect that non-Gaussian interference has on the performance of a receiver that has been optimized for Gaussian noise is determined. The factors affecting the choice of modulation scheme so as to minimize the deterimental effects of non-Gaussian noise are then discussed. In the second part, a new theoretical model of impulsive noise that fits well with the observed statistics of noise in radio channels below 100 MHz has been developed. This empirical noise model is applied to the detection of known signals in the presence of noise to determine the optimal receiver structure. The performance of such a detector has been assessed and is found to depend on the signal shape, the time-bandwidth product, as well as the signal-to-noise ratio. The optimal signal to minimize the probability of error of; the detector is determined. Attention is then turned to the problem of threshold detection. Detector structure, large sample performance and robustness against errors in the detector parameters are examined. Finally, estimators of such parameters as. the occurrence of an impulse and the parameters in an empirical noise model are developed for the case of an adaptive system with slowly varying conditions.

Coalescence of secondary dispersions

A study has been made of the coalescence of secondary dispersions in beds of woven meshes. The variables investigated were superficial velocity, bed depth, mesh geometry and fibre material; the effects of presoaking the bed in the dispersed phase before operation were also considered. Equipment was design~d to generate a 0.1% phase ratio toluene in water dispersion whose mean drop size was determined using a Coulter Counter. The coalesced drops were sized by photography and a novel holographic technique was developed to evaluate the mean diameter of the effluent secondary drops. Previous models describing single phase flow in porous media are reviewed and it was found that the experimental data obtained in this study is best represented by Keller's equation which is based on a physical model similar to the internal structure of the meshes. Statistical analysis of two phase data produced a correlation, for each mesh tested, relating the pressure drop to superficial velocity and bed depth. The flow parameter evaluated from the single phase model is incorporated into a theoretical comparison of drop capture mechanisms which indicated that direct and indirect interception are predominant. The resulting equation for drop capture efficiericy is used to predict the initial, local drop capture rate in a coalescer. A mathematical description of the saturation profiles was formulated and verified by average saturation data. Based 6n the Blake-Kozeny equation, an expression is derived analytically to predict the two phase pressure drop using the parameters which characterise the saturation profiles. By specifying the local saturation at the inlet face for a given velocity, good agreement between experimental pressure drop data and the model predictions was obtained.

AIDS: structure, replication and spread of the AIDS virus

AIDS (Acquired Immune Deficiency Syndrome)was first, described as a new disease of humans in 1981. The origins of the disease are controversial. AIDS is caused by a retrovirus, a type of virus which rarely attacks human cells. The first virus of this type recorded in humans is reponsible for a type of leukaemia and was identified in 1978. AIDS is thus the third type of human retrovirus to be discovered and hence, is referred to as T-lymphotrophic virus III (HTLV-III). For viruses to replicate, they have to invade a host cell which in this case is a T4-lymphocyte, a type of white blood cell that regulates the immune system. The problems of the disease result directly from the death of these cells. As a consequence, the immune system is compromised leading to a number of opportunistic secondary infections and other disorders.

A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot-protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins.

Antibody detection of Burkholderia pseudommallei and Burkholderia mallei

Monoclonal and polyclonaI antibodies have been produced for use in immunological assays for the detection of Burkholderia pseudomallei and Burkholderia mallei. Monoclonal antibodies recognising a high molecular weight polysaccharide material found in some strains of both species have been shown to be effective in recognising B. pseudomallei and B. mallei and distinguishing them from other organisms. The high molecular weight polysaccharide material is thought to be the capsule of B. pseudomallei and B. mallei and may have important links with virulence. B. pseudomallei and B. mallei are known to be closely related, sharing many epitopes, but antigenic variation has been demonstrated within both the species. The lipopolysaccharide from strains of B. pseudomal/ei and B. mallei has been isolated and the silver stain profiles found to be visually very similar. A monoclonal antibody raised to B. mallei LPS has been found to recognise both B. mallei and B. pseudomallei strains. However, in a small number of B. pseudomallei strains a visually atypical LPS profile has been demonstrated. A monoclonal ant ibody rai sed against this atypical LPS showed no recognition of the typical LPS profile of either B. mallei or B. pseudomallei. This atypical LPS structure has not been reported and may be immunologically distinct from the typical LPS. Molecular biology and antibody engineering techniques have been used in an attempt to produce single-chain antibody fragments reactive to B. pseudomallei. Sequencing of one of the single-chain antibody fragments produced showed high homology with murine immunoglobulin genes, but none of the single-chain antibody fragments were found to be specific to B. pselldomallei.

The combination of smart hydrogels and fibre optic sensor technology for analyte detection

The subject of investigation of the present research is the use of smart hydrogels with fibre optic sensor technology. The aim was to develop a costeffective sensor platform for the detection of water in hydrocarbon media, and of dissolved inorganic analytes, namely potassium, calcium and aluminium. The fibre optic sensors in this work depend upon the use of hydrogels to either entrap chemotropic agents or to respond to external environmental changes, by changing their inherent properties, such as refractive index (RI). A review of current fibre optic technology for sensing outlined that the main principles utilised are either the measurement of signal loss or a change in wavelength of the light transmitted through the system. The signal loss principle relies on changing the conditions required for total internal reflection to occur. Hydrogels are cross-linked polymer networks that swell but do not dissolve in aqueous environments. Smart hydrogels are synthetic materials that exhibit additional properties to those inherent in their structure. In order to control the non-inherent properties, the hydrogels were fabricated with the addition of chemotropic agents. For the detection of water, hydrogels of low refractive index were synthesized using fluorinated monomers. Sulfonated monomers were used for their extreme hydrophilicity as a means of water sensing through an RI change. To enhance the sensing capability of the hydrogel, chemotropic agents, such as pH indicators and cobalt salts, were used. The system comprises of the smart hydrogel coated onto an exposed section of the fibre optic core, connected to the interrogation system measuring the difference in the signal. Information obtained was analysed using a purpose designed software. The developed sensor platform showed that an increase in the target species caused an increase in the signal lost from the sensor system, allowing for a detection of the target species. The system has potential applications in areas such as clinical point of care, water detection in fuels and the detection of dissolved ions in the water industry.

Early visual detection of oriented line targets: spatial characteristics,and reference frames of early visual orientation-processing

The orientations of lines and edges are important in defining the structure of the visual environment, and observers can detect differences in line orientation within the first few hundred milliseconds of scene viewing. The present work is a psychophysical investigation of the mechanisms of early visual orientation-processing. In experiments with briefly presented displays of line elements, observers indicated whether all the elements were uniformly oriented or whether a uniquely oriented target was present among uniformly oriented nontargets. The minimum difference between nontarget and target orientations that was required for effective target-detection (the orientation increment threshold) varied little with the number of elements and their spatial density, but the percentage of correct responses in detection of a large orientation-difference increased with increasing element density. The differing variations with element density of thresholds and percent-correct scores may indicate the operation of more than one mechanism in early visual orientation-processIng. Reducing element length caused threshold to increase with increasing number of elements, showing that the effectiveness of rapid, spatially parallel orientation-processing depends on element length. Orientational anisotropy in line-target detection has been reported previously: a coarse periodic variation and some finer variations in orientation increment threshold with nontarget orientation have been found. In the present work, the prominence of the coarse variation in relation to finer variations decreased with increasing effective viewing duration, as if the operation of coarse orientation-processing mechanisms precedes the operation of finer ones. Orientational anisotropy was prominent even when observers lay horizontally and viewed displays by looking upwards through a black cylinder that excluded all possible visual references for orientation. So, gravitational and visual cues are not essential to the definition of an orientational reference frame for early vision, and such a reference can be well defined by retinocentric neural coding, awareness of body-axis orientation, or both.

Aptamer-based surface plasmon sensor for thrombin detection

A series of surface plasmonic fibre devices were fabricated using multiple coatings deposited on a lapped section of a single mode fibre and post-fabrication UV laser irradiation processing with a phase mask, producing a surface relief grating structure. These devices showed high spectral sensitivity in the aqueous index regime ranging up to 4000 nm/RIU for wavelength and 800 dB/RIU for intensity. The devices were then coated with human thrombin binding aptamer. Several concentrations of thrombin in buffer solution were made, ranging from 1nM to 1µM. All the concentrations were detectable by the devices demonstrating that sub-nM concentrations may be monitored.