Search results for "denaturation"

Heparan sulfate proteoglycans interact exclusively with conformationally intact HPV L1 assemblies: basis for a virus-like particle ELISA.

2004

In this article, we demonstrate that interaction of human papillomavirus-like particles (HPV-VLPs) with the putative glucosaminoglycan binding receptor is strictly dependent on conformational integrity. Such conformations are present on VLPs and capsomeres but not on monomers of the major capsid protein, L1, confirming reports that capsomeres can induce virus-neutralizing antibodies. Furthermore, we show the suitability of this specific interaction for development of VLP-based enzyme-linked immunosorbent assays (ELISAs), using heparin for indirect coupling of VLPs to microtiter plates, which may add an intrinsic quality control. This avoids presentation of linear, often highly cross-reactiv…

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex.

2002

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. …

Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.

1998

The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar …

A reduction of protein specific motions in co-ligated myoglobin embedded in a trehalose glass

2000

Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding

2020

International audience; Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the protein main chain. Because chemical shift (CS) measured by NMR spectroscopy is extremely sensitive to the local atomic environment, CS has served as a local probe of thermal unfolding of proteins by varying the position of the atomic isotope along the amino-acid sequence. The variation of the CS of each C(alpha) atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We…

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

2009

The major light-harvesting chlorophyll a / b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin dist…

Protein Thermal Denaturation and Matrix Glass Transition in Different Protein−Trehalose−Water Systems

2011

Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose…

Denaturation via Surfactants Changes Composition of Protein Corona

2018

The use of nanocarriers as drug delivery vehicles brings them into contact with blood plasma proteins. Polymeric nanocarriers require some sort of surfactant to ensure colloidal stability. Formation of the protein corona is therefore determined not only by the intrinsic properties of the nanocarrier itself but also by the accompanying surfactant. Although it is well-known that surfactants have an impact on protein structure, only few studies were conducted on the specific effect of surfactants on the composition of protein corona of nanocarriers. Therefore, we analyzed the composition of the protein corona on "stealth" nanoparticles with additional surfactant (cetyltrimethylammonium chlorid…

Processing requirements for the recognition of insulin fragments by murine T cells.

1988

In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A…

Template-Directed Protein Folding into a Metastable State of Increased Activity

1995

The principal objective of this work was to distinguish between kinetic and thermodynamic reaction control in protein folding. The deleterious effects of a specific mutation on spontaneous refolding competence were analyzed for this purpose. A Bowman-Birk-type proteinase inhibitor of trypsin and chymotrypsin was selected as a double-headed model protein to facilitate the detection of functional irregularities by the use of functional assays. The parent protein spontaneously folds into a single, fully active and thermodynamically stable state in a redox buffer after reduction/denaturation. By contrast, the properties of a P'1Ser--Pro variant in the trypsin-reactive subdomain differ before an…