A controlled co-assembly approach to tune temperature responsiveness of biomimetic proteins.

The controlled co-assembly of biomacromolecules through tuneable interactions offers a simple and fascinating opportunity to assemble multiple molecules into a single entity with enhanced complexity and unique properties. Herein, our study presents a dynamic co-assembled system using the multistimuli responsive intrinsically disordered protein Rec1-resilin and the adhesive hydrophilic protein silk sericin (SS). We utilized advanced characterization techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and small/ultra-small angle neutron scattering (SANS/USANS) to elucidate the detailed co-assembly behavior of the system and its evolution over time and temperature.

Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes.

Crowder-directed interactions and conformational dynamics in multistimuli-responsive intrinsically disordered protein.

The consequences of crowding on the dynamic conformational ensembles of intrinsically disordered proteins (IDPs) remain unresolved because of their ultrafast motion. Here, we report crowder-induced interactions and conformational dynamics of a prototypical multistimuli-responsive IDP, Rec1-resilin. The effects of a range of crowders of varying sizes, forms, topologies, and concentrations were examined using spectroscopic, spectrofluorimetric, and contrast-matching small- and ultrasmall-angle neutron scattering investigation.

Deuteration for biological SANS: Case studies, success and challenges in chemistry and biology.

Small angle neutron scattering is a powerful complementary technique in structural biology. It generally requires, or benefits from, deuteration to achieve its unique potentials. Molecular deuteration has become a mature expertise, with deuteration facilities located worldwide to support access to the technique for a wide breadth of structural biology and life sciences.

The Aggregation of αB-Crystallin under Crowding Conditions Is Prevented by αA-Crystallin: Implications for α-Crystallin Stability and Lens Transparency.

One of the most crowded biological environments is the eye lens which contains a high concentration of crystallin proteins. The molecular chaperones αB-crystallin (αBc) with its lens partner αA-crystallin (αAc) prevent deleterious crystallin aggregation and cataract formation. However, some forms of cataract are associated with structural alteration and dysfunction of αBc.

Conformational selection of the intrinsically disordered plant stress protein COR15A in response to solution osmolarity - an X-ray and light scattering study.

The plant stress protein COR15A stabilizes chloroplast membranes during freezing. COR15A is an intrinsically disordered protein (IDP) in aqueous solution, but acquires an α-helical structure during dehydration or the increase of solution osmolarity. We have used small- and wide-angle X-ray scattering (SAXS/WAXS) combined with static and dynamic light scattering (SLS/DLS) to investigate the structural and hydrodynamic properties of COR15A in response to increasing solution osmolarity.

Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation.

We have simplified the structural heterogeneity of protein-polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (βLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the βLgA-OGA interaction is critically dependent on the length of the oligosaccharide.

Robust high-yield methodologies for (2)H and (2)H/(15)N/(13)C labeling of proteins for structural investigations using neutron scattering and NMR.

We have developed a method that has proven highly reliable for the deuteration and triple labeling ((2)H/(15)N/(13)C) of a broad range of proteins by recombinant expression in Escherichia coli BL21. Typical biomass yields are 40-80g/L wet weight, yielding 50-500mg/L purified protein. This method uses a simple, relatively inexpensive defined medium, and routinely results in a high-yield expression without need for optimization.

Interrogating protonated/deuterated fibronectin fragment layers adsorbed to titania by neutron reflectivity and their concomitant control over cell adhesion.

The fibronectin fragment, 9th-10th-type III domains (FIII9-10), mediates cell attachment and spreading and is commonly investigated as a bioadhesive interface for implant materials such as titania (TiO2). How the extent of the cell attachment-spreading response is related to the nature of the adsorbed protein layer is largely unknown. Here, the layer thickness and surface fraction of two FIII9-10 mutants (both protonated and deuterated) adsorbed to TiO2 were determined over concentrations used in cell adhesion assays.

Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study.

The 140 residue intrinsically disordered protein α-synuclein (α-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in α-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu2+) or an anti-fibril organic platinum (Pt) complex.

Guanidine hydrochloride denaturation of dopamine-induced α-synuclein oligomers: a small-angle X-ray scattering study.

Alpha-synuclein (α-syn) forms the amyloid-containing Lewy bodies found in the brain in Parkinson's disease. The neurotransmitter dopamine (DA) reacts with α-syn to form SDS-resistant soluble, non-amyloid, and melanin-containing oligomers. Their toxicity is debated, as is the nature of their structure and their relation to amyloid-forming conformers of α-syn.

Monitoring the interaction between β2-microglobulin and the molecular chaperone αB-crystallin by NMR and mass spectrometry: αB-crystallin dissociates β2-microglobulin oligomers.

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β2-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m.

The chaperone activity of α-synuclein: Utilizing deletion mutants to map its interaction with target proteins.

α-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, α-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. α-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro.

The quaternary organization and dynamics of the molecular chaperone HSP26 are thermally regulated.

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature.

A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, αA- and αB-crystallin.

The principal lens proteins αA- and αB-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, αA- and αB-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. αB-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization.

The structure of dopamine induced alpha-synuclein oligomers.

Inclusions of aggregated alpha-synuclein (alpha-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson's disease (PD). In vitro, alpha-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of alpha-syn oligomers formed in the presence of DA.

Small-angle X-ray scattering study of the effect of pH and salts on 11S soy glycinin in the freeze-dried powder and solution states.

The nanostructures from powders of native protein, glycinin, and corresponding solutions from which the powders have been formed, have been studied as a function of pH and 1 M salts using small-angle X-ray scattering. All powders showed Porod scattering with the exception of that prepared from the solution close to pI which displayed fractal behavior. Well-defined Bragg peaks in the powder scattering at pH 5, pH 7, and 1 M NaCl indicate the presence of long-range order.

PAMAM dendrimers as potential agents against fibrillation of alpha-synuclein, a Parkinson's disease-related protein.

The effect of PAMAM dendrimers (generations G3, G4 and G5) on the fibrillation of alpha-synuclein was examined by fluorescence and CD spectroscopy, TEM and SANS. PAMAM dendrimers inhibited fibrillation of alpha-synuclein and this effect increased both with generation number and PAMAM concentration. SANS showed structural changes in the formed aggregates of alpha-synuclein--from cylindrical to dense three-dimensional ones--as the PAMAM concentration increased, on account of the inhibitory effect.

Monitoring the prevention of amyloid fibril formation by alpha-crystallin. Temperature dependence and the nature of the aggregating species.

The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin.

The effect of dextran on subunit exchange of the molecular chaperone alphaA-crystallin.

Alpha-crystallin, a member of small heat shock protein (sHsp) family, is comprised of alphaA and alphaB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The alphaA-crystallin homopolymer consists of 30-40 subunits that are undergoing dynamic exchange. In vivo, alpha-crystallin elicits its chaperone action in a crowded cellular environment (e.

Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS- and beta-casein.

Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e.

R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable.

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin.

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity.

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD.

Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function?

Small heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly.

Crystal structure of venus, a yellow fluorescent protein with improved maturation and reduced environmental sensitivity.

Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP).