Malleability of the folding mechanism of the outer membrane protein PagP: parallel pathways and the effect of membrane elasticity.

Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the folding mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the folding mechanism of the bacterial outer membrane protein PagP. The folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the folding and insertion of the protein into the membrane.

Conformational properties of the unfolded state of Im7 in nondenaturing conditions.

The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate.

Phosphorylation as a tool to modulate aggregation propensity and to predict fibril architecture.

Despite the importance of post-translational modifications in controlling the solubility and conformational properties of proteins and peptides, precisely how the aggregation propensity of different peptide sequences is modulated by chemical modification remains unclear. Here we have investigated the effect of phosphorylation on the aggregation propensity of a 13-residue synthetic peptide incorporating one or more phosphate groups at seven different sites at various pH values. Fibril formation was shown to be inhibited when a single phosphate group was introduced at all seven locations in the peptide sequence at pH 7.

Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils.

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β(2)-microglobulin (β(2)m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood.

Second order rate constants of donor-strand exchange reveal individual amino acid residues important in determining the subunit specificity of pilus biogenesis.

P pili are hair-like adhesive structures that are assembled on the outer membrane (OM) of uropathogenic Escherichia coli by the chaperone-usher pathway. In this pathway, chaperone-subunit complexes are formed in the periplasm and targeted to an OM assembly platform, the usher. Pilus subunits display a large groove caused by a missing β-strand which, in the chaperone-subunit complex, is provided by the chaperone.

Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly.

β(2)-Microglobulin is a 99-residue protein with a propensity to form amyloid-like fibrils in vitro which exhibit distinct morphologies dependent on the solution conditions employed. Here we have used ion mobility spectrometry-mass spectrometry to characterize the oligomeric species detected during the formation of worm-like fibrils of β(2)-microglobulin at pH 3.6.

Ligand binding to distinct states diverts aggregation of an amyloid-forming protein.

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β(2) microglobulin (β(2)m) fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism.

A diversity of assembly mechanisms of a generic amyloid fold.

Protein misfolding and amyloid assembly have long been recognized as being responsible for many devastating human diseases. Recent findings indicate that amyloid assemblies may facilitate crucial biological processes from bacteria to mammals. This review focuses on the mechanistic understanding of amyloid formation, including the transformation of initially innocuous proteins into oligomers and fibrils.

Women and firesetting: a qualitative analysis of context, meaning, and development.

Objectives: To explore subjective experiences of women in secure services of their firesetting behaviour, its personal meaning and the factors that contributed to its development.

Design: An interview-based study using Interpretative Phenomenological Analysis.

Methods: Interviews were carried out with nine women, in medium secure services, with histories of firesetting.

Improving health outcomes in young people - a holistic, team based approach.

Background: Young people aged 12-25 years are poorly serviced by current models of healthcare; they are under represented in Medicare data and are poor seekers of healthcare. However, the majority of mental health problems commence during this age span, significant sexual health issues arise, and there is poor compliance with treatment for chronic disease.

Objective: This article describes a holistic, multisector primary healthcare delivery model which may provide a way forward to improve both access and outcomes for young people.

Understanding the complex mechanisms of β2-microglobulin amyloid assembly.

Several protein misfolding diseases are associated with the conversion of native proteins into ordered protein aggregates known as amyloid. Studies of amyloid assemblies have indicated that non-native proteins are responsible for initiating aggregation in vitro and in vivo. Despite the importance of these species for understanding amyloid disease, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they aggregate remain elusive.

Systematic examination of polymorphism in amyloid fibrils by molecular-dynamics simulation.

Amyloid fibrils often exhibit polymorphism. Polymorphs are formed when proteins or peptides with identical sequences self-assemble into fibrils containing substantially different arrangements of the β-strands. We used atomistic molecular-dynamics simulation to examine the thermodynamic stability of a amyloid fibrils in different polymorphic forms by performing a systematic investigation of sequence and symmetry space for a series of peptides with a range of physicochemical properties.

Dissecting key residues in folding and stability of the bacterial immunity protein 7.

The small four-helix immunity protein, Im7, has previously been shown to fold via a compact intermediate containing three of the four native helices. The short, six-residue helix III only docks onto the developing Im7 structure after the rate-limiting second transition state has been traversed. Previous work demonstrated that mutation of the helix III sequence can be used to trap the protein in the on-pathway intermediate ensemble at equilibrium.

Conformational conversion during amyloid formation at atomic resolution.

Numerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ΔN6, a truncation variant of the naturally amyloidogenic protein β(2)-microglobulin (β(2)m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution.

Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).

Single molecule Förster resonance energy transfer (FRET) experiments are a versatile method for investigating the conformational distributions and dynamics of biological macromolecules. In a common type of experiment, the fluorescence bursts from individual molecules freely diffusing in solution are detected as they pass through the observation volume of a confocal microscope. Correlation analysis of the fluorescence bursts shows that under typical experimental conditions, for time scales up to several tens of milliseconds, the probability for a molecule to return to the confocal volume is greater than the probability of a new molecule being detected.

Successful pregnancy following mid-trimester evacuation through a transabdominal cervical cerclage.

Preterm birth remains a major challenge in modern obstetrics and is increasing, even among low-risk primiparous women. Very few interventions have made a positive impact on outcome although cervical cerclage appears to benefit some women. Transabdominal cervical cerclage can be highly successful and should be considered in women with previous failed transvaginal cerclage, but requires operative abdominal delivery as it cannot be removed.

Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation.

N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc).

A structural model for apolipoprotein C-II amyloid fibrils: experimental characterization and molecular dynamics simulations.

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets.

Intermolecular alignment in β2-microglobulin amyloid fibrils.

The deposition of amyloid-like fibrils, composed primarily of the 99-residue protein β2-microglobulin (β2m), is one of the characteristic symptoms of dialysis-related amyloidosis. Fibrils formed in vitro at low pH and low salt concentration share many properties with the disease related fibrils and have been extensively studied by a number of biochemical and biophysical methods. These fibrils contain a significant β-sheet core and have a complex cryoEM electron density profile.

The oligomeric state and arrangement of the active bacterial translocon.

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods.

How does mindfulness-based cognitive therapy work?

Mindfulness-based cognitive therapy (MBCT) is an efficacious psychosocial intervention for recurrent depression (Kuyken et al., 2008; Ma & Teasdale, 2004; Teasdale et al., 2000).

Magic angle spinning NMR analysis of beta2-microglobulin amyloid fibrils in two distinct morphologies.

Beta(2)-microglobulin (beta(2)m) is the major structural component of amyloid fibrils deposited in a condition known as dialysis-related amyloidosis. Despite numerous studies that have elucidated important aspects of the fibril formation process in vitro, and a magic angle spinning (MAS) NMR study of the fibrils formed by a small peptide fragment, structural details of beta(2)m fibrils formed by the full-length 99-residue protein are largely unknown. Here, we present a site-specific MAS NMR analysis of fibrils formed by the full-length beta(2)m protein and compare spectra of fibrils prepared under two different conditions.

Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry.

The key to understanding amyloid disease is the characterization of oligomeric species formed during the early stages of fibril assembly. Here we have used electrospray ionisation-ion mobility spectrometry-mass spectrometry to identify and structurally characterize the oligomers formed during amyloid assembly from beta(2)-microglobulin (beta(2)m). Beta(2)m oligomers are shown to have collision cross-sections consistent with monomeric units arranged in elongated assemblies prior to fibril formation.