Identification of membrane spanning beta strands in bacterial porins

The membrane assembly of outer membrane proteins is more complex than that of transmembrane helical proteins owing to the intervention of many charged and polar residues in the membrane. Accordingly, the predictive accuracy of transmembrane beta strands is considerably lower than that of transmembrane alpha helices. In this paper we develop a set of conformational parameters for membrane spanning beta strands. We formulate an algorithm to predict the transmembrane beta strands in the family of bacterial porins based on the conformational parameters and surrounding hydrophobicities of amino acid residues. A Fortran program has been developed which takes the amino acid sequence as the input file and gives the predicted transmembrane beta strand as output. The present method predicts at an accuracy level of 82% for all the bacterial porins considered.      

Kinetics and thermodynamics of amyloid fibril assembly

With some exceptions, amyloids appear to be accidental aggregated structures whose formation was not selected for in molecular evolution. Despite this, amyloid fibrils are in many respects surprisingly well-behaved molecules. For example, Huntington's disease-related polyglutamine sequences aggregate via a relatively simple nucleated growth polymerization mechanism. In addition, the Alzheimer's plaque protein Abeta has been shown to undergo reversible amyloid fibril formation to a position of dynamic equilibrium such that reaction thermodynamics can be quantified. Studies of these well-behaved amyloid systems are allowing us to peer more deeply into the process and products of off-pathway misfolding and aggregation.     

Designing proteins to crystallize through {beta}-strand pairing

Inherent difficulties in growing protein crystals are majorconcerns within structural biology and particularly in structuralproteomics. Here, we describe a novel approach of engineeringtarget proteins by surface mutagenesis to increase the oddsof crystallizing the molecules. To this end, we have exploitedour recent triad-hypothesis using proteins with crystallographicallydefined ß-structures as the principal models. Crystalpacking analyses of 182 protein structures belonging to 21 differentsuperfamilies implied that the propensities to crystallize couldbe engineered into target proteins by replacing short segments,5–6 residues, of their ß-strands with ‘cassettes’of suitable packing motifs. These packing motifs will generatespecific crystal packing interactions that promote crystallization.Key features of the primary and tertiary structures of suchpacking motifs have been identified for immunoglobulins. Further,packing motifs have been engineered successfully into six modelantibodies without disturbing their capabilities to be produced,their immunoreactivity and their overall structure. Preliminarycrystallization analyses have also been performed. Taken together,the procedures outline a rational protocol for crystallizingproteins by surface mutagenesis. The importance of these findingsis discussed in relation to the crystallization of proteinsin general.     

Amide inequivalence in the fibrillar assembly of islet amyloid polypeptide

Amyloid fibers are aggregated, yet highly ordered, beta-sheet-rich assemblies of misfolded proteins. Order is established in such systems following profiles indicative of nucleation-dependent assembly. Nucleation dependence suggests that specific interactions, such as long-range contacts and/or strand registration, are critical to establishing initial fiber structure. Here, we show that amino acids at selected positions participate in key interactions that modulate the pathway of amyloid fiber formation by the hormone, islet amyloid polypeptide (IAPP). Specifically, we investigated the role of amide side-chain interactions in the process of IAPP assembly. We mutated five of the asparagine side chains in IAPP and assessed their effects on the kinetics of assembly. We find that the asparagine amide side chains strongly dictate the ability of IAPP to form fibers. In particular, the elimination of two specific asparagines results in near and total loss of amyloid, respectively. Interestingly, the two asparagines are located in a recently identified domain with alpha-helical bias. These sensitivities are unusual for IAPP, as IAPP is generally tolerant to mutation. Here, we demonstrate this mutational tolerance by assessing 10 alterations at five distinct sites. In all cases, the constructs form fibers on timescales perturbed by less than a factor of two compared with wild-type protein. These findings indicate the presence of key specific interactions that are the determinants of IAPP amyloid formation.     

Elucidating amyloid beta-protein folding and assembly: A multidisciplinary approach

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid beta-protein, the key pathogenetic agent in Alzheimer's disease.     

Stranded in isolation: structural role of isolated extended strands in proteins

Reasons for the formation of extended-strands (E-strands) inproteins are often associated with the formation of ß-sheets.However E-strands, not part of ß-sheets, commonlyoccur in proteins. This raises questions about the structuralrole and stability of such isolated E-strands. Using a datasetof 250 largely non-homologous and high-resolution (<2 Å)crystal structures of proteins, we have identified 518 isolatedE-strands from 187 proteins. The two most distinguishing featuresof isolated E-strands from ß-strands in ß-sheetsare the high preponderance of prolyl residues occuring in isolatedE-strands and their high exposure to the surroundings. Removalof regions with polyproline conformation from the dataset didnot significantly reduce the propensity of prolyl residues tooccur in isolated E-strands. Isolated E-strands are often characterizedby their main-chain amide and carbonyl groups involved in hydrogenbonding with polar side chains or water. They are often flankedby irregular loop structures and are less well conserved, thanß-sheet forming ß-strands, among homologousprotein structures. It is suggested that isolated ß-strandshave many characteristics of loop segments but with repetitive(     

The role of the sequence extensions in {beta}-crystallin assembly

The modular construction of the eye lens ß-crystallinsmakes them good candidates for protein engineering to ascertainthe rules of assembly of oligomers. X-ray studies have shownthat although the polypeptide chains of ßB2-crystallinand -crystallins fold to form similar N- and C-terminal domains,the conformation of the connecting peptides are such that the-crystallins are monomers and the ß-crystallin isa dimer. Unlike -crystallins, the numerous -crystallins haveextensions of variable sequence from the globular domains. Wehave tested the effect of removing the N- and C-terminal extensionsfrom rat ßB2-crystallin using a bacterial expressionsystem. Abundant proteins were produced in Escherichia coliusing the pET or pQE vectors. Full-length and truncated proteinswere purified and checked for refolding using circular dichroism.Sizing of the truncated proteins using gel filtration chroma-tographyshowed that the absence of either the N- or C-terminal extensiondoes not affect dimerization of ßB2-crystallin.     

Length preferences and periodicity in {beta}-strands. Antiparallel edge {beta}-sheets are more likely to finish in non-hydrogen bonded rings

We analysed the length distributions of different types of ß-strandin a high resolution, non-homologous set of 500 protein structures,finding differences in their mean lengths. Antiparallel edgestrands in strand–turn–strand motifs show a preferencefor an even number of residues. This propensity is enhancedif the length is corrected for ß-bulges, which insertan extra residue into the strand. Residues in antiparallel edgeß-strands alternate between being in hydrogen bondedand non-hydrogen bonded rings. Antiparallel edges with an evennumber of residues are more likely to have their final ßresidue in a non-hydrogen bonded ring. This suggests that non-hydrogenbonded rings are intrinsically more stable than hydrogen bondedrings, perhaps because its side chain packing is closer. Therefore,we suggest that a simple way to increase ß-hairpinstability, or the stability of an antiparallel edge strand,is to have a non-hydrogen bonded ring at the end of the strand. Received June 19, 2003; revised October 25, 2003; accepted November 7, 2003     

Plastic deformation of ZnO thin films through edge and screw dislocation movements

Columnar wurtzite grains were formed in sputtered ZnO thin films deposited on a plastic polyethylene terephthalate substrate. Selected-area diffraction patterns reveal that the columnar grains in the sputtered films present two preferred growth planes, namely, the basal (0002) and prismatic (100) growth planes. The diffraction patterns obtained also confirm that the microstructure of sputtered indium tin oxide thin films is amorphous in nature. Tensile tests indicate that the fracture strain of the ZnO thin film occurs between 1.73% and 2.14%, while the fracture strain of the indium tin oxide thin film occurs between 0.24% and 0.67%. Thus, the fracture toughness of the sputtered ZnO thin film is greater than that of the sputtered indium tin oxide thin film. High-resolution transmission electron microscopic images demonstrate that edge and screw dislocations could be identified in the sputtered ZnO thin films. Moreover, edge and screw dislocation movements may, respectively, be observed in the basal- and prismatic-oriented ZnO columnar grains of the sputtered ZnO thin films. Our results indicate that movements of the edge and screw dislocations in the basal- and prismatic-oriented ZnO columnar grains account for the plastic deformation of the investigated ZnO thin films under tensile stress.     

Monitoring roughness and edge shape on semiconductors through multiresolution and multivariate image analysis

Photolithography is one of the most important processes in the production of integrated circuits. Usually, attentive inspections are required after this process, but are limited to the measurement of some physical parameters such as the critical dimension and the line edge roughness. In this paper, a novel multiresolution multivariate technique is presented to identify the abnormalities on the surface of a photolithographed device and the location of defects in a sensitive fashion by comparing it to a reference optimum, and generating fast, meaningful and reliable information. After analyzing the semiconductor surface image in different levels of resolutions via wavelet decomposition, the application of multivariate statistical monitoring tools allows the in‐depth examination of the imprinted features of the product. A two level nested PCA model is used for surface roughness monitoring, while a new strategy based on “spatial moving window” PCA is proposed to analyze the shape of the patterned surface. The effectiveness of the proposed approach is tested in the case of semiconductor surface SEM images after the photolithography process. The approach is general and can be applied also to inspect a product through different types of images, different phases of the same production systems, or different processes. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

A backbone-reversed all-{beta} polypeptide (retro-CspA) folds and assembles into amyloid nanofibres

The backbone-reversed or ‘retro’, form of a modelall-ß-sheet protein, Escherichia coli CspA, was producedfrom a synthetic gene in E.coli in fusion with an N-terminalaffinity tag. Following purification under denaturing conditionsand dialysis-based removal of urea, the protein was found tofold into a soluble, poorly structured multimer. Upon concentration,this state readily transformed into amyloid nanofibres. CongoRed-binding amorphous forms were also observed. Since a ß-sheet-formingsequence is expected to retain high ß-sheet-formingpropensity even after backbone reversal and given the fact thatfolding of retro-CspA occurs only to a poorly structured form,we conclude that the increase effected in protein concentrationmay be responsible for the formation of intermolecular ß-sheets,facilitating the bleeding away of the protein’s conformationalequilibrium into aggregates that generate well-formed fibres.Since every molecule in these fibres contains a peptide tagfor binding Ni²⁺, the fibres may provide a template for depositionof nickel to generate novel materials. Received April 1, 2003; revised October 27, 2003; accepted October 30, 2003     

A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease

We provide a validated and rapid protocol for the solubilization of amyloid β-peptide (Aβ). This procedure involves sequential solubilization using structure-breaking organic solvents hexafluoroisopropanol and DMSO followed by column purification. The low solubility and tendency of Aβ to aggregate considerably impede the in vitro handling and biophysical or biological investigation of Aβ, despite the interest in this peptide because of its implication in Alzheimer's disease. The main advantage of the proposed protocol over others is that it results in standardized aggregate-free Aβ peptide samples that are biocompatible for cell culture studies and yield reproducible aggregation kinetics and cytotoxicities. This three-step protocol also enables the co-solubilization of the longer Aβ42 variant with Aβ40 in ratios relevant to Alzheimer's disease.     

Superficial degradation evaluated through color change in weathered orange LLDPE

We report the evaluation of superficial degradation of orange and colorless linear low density polyethylene samples exposed to weather. Color change and UV–VIS absorption were measured on samples exposed to the weather conditions of the city of Aguascalientes, Mexico, for more than a year. The color change was calculated in CIELAB units. Color change and absorption presented an exponential decay with time for orange samples, whereas they remained constant for colorless samples. No degradation products from the polymer matrix were revealed by UV–VIS spectroscopy, and apart from color change, no other degradation was noticed. © 2009 Wiley Periodicals, Inc. Col Res Appl, 34, 458–463, 2009