The covalent attachment of a bacterial-derived capsular polysaccharide to protein is of critical importance in transforming the polysaccharide from an antigen with limited immunogenicity in infants ...and older adults to an antigen that can prevent potentially fatal disease. For a polysaccharide-protein conjugate vaccine (PCV) candidate to be successful, it must be sufficiently stable. Chemical breakage of carbohydrate bonds in the polysaccharide may result in the reduction of “conjugate dose” and could negatively impact immunogenicity and the ability of the vaccine to prime for memory responses. Therefore, development of analytical tools to monitor the integrity of a polysaccharide-protein conjugate (glycoconjugate) vaccine is of practical significance. In this work, reducing SDS-PAGE, Intrinsic Protein Fluorescence Spectroscopy (IPFS), Differential Scanning Fluorimetry (DSF) were evaluated methods to study the impact of time, temperature, and formulation composition on the stability of a glycoconjugate vaccine prepared by multisite coupling of polysaccharide to a carrier protein. In addition, an automated capillary Western system was also evaluated to study the impact of storage on glycoconjugate vaccine stability. Two streptococcus pneumoniae polysaccharide-protein conjugates (serotype 3 and serotype 19A) were chosen to examine their physicochemical stability when formulated as a single antigen vaccine. While all methods require only a small amount of test article and can test multiple samples per assay run, automated capillary Western has the additional advantage of being highly sensitive even at low concentrations in complex vaccine formulations that contain aluminum adjuvant and multiple antigens. Results suggest that automated capillary Western is stability-indicating and may be an effective analytical technology tool for the formulation development of a multivalent glycoconjugate vaccine.
Several spectral methods (UV absorbance, fluorescent and FTIR spectroscopy) were applied to reveal the alterations in the secondary and tertiary structures of human serum albumin (HSA) in water ...solutions under various conditions (pH, protein concentration, ethanol and n-propanol addition). The structural transitions well known for defatted HSA at 1.5 < pH < 13 are observed for HSA in a complex with long-chain fatty acids in the molar ratio about 1:1. The changes of HSA ζ-potential during the protein isomerizations induced by the variation of pH are traced. It is shown that HSA does not completely lose its secondary structure at extreme alkaline or acidic solutions, which indicates that at these conditions HSA takes the “molten globule” conformation. A comparison of aggregation processes of HSA and ovalbumin in neutral water and 0.15 M NaCl solutions reveals that OVA aggregation is preceded by a partially denaturated state of the protein, whereas an intermediates of HSA aggregates are close to the native state of the protein. The aliphatic alcohols disturb the tertiary structure of HSA, but stabilize its secondary structure. This effect increases with the rise of the alcohol hydrophobicity.
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•Changes in the secondary and tertiary structures of human serum albumin (HSA) caused by the ionization of amino acids side chains were traced.•Ovalbumin aggregation is preceded by a partially denaturated state of the protein. An intermediates of HSA aggregates are close to the native state of the protein.•The aliphatic alcohols disturb the tertiary structure of HSA, but stabilize its secondary structure.
The observables associated with protein intrinsic fluorescence – spectra, time decays, anisotropies – offer opportunities to monitor in real time and non‐invasively a protein‘s functional form and ...its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.
A matter of form: The active and the inactive functional forms of human thymidylate synthase, a long‐known anticancer target, exhibit different intrinsic fluorescence spectra, quantum yields, lifetime profiles and anisotropies. These differences are connected to the conformational changes occurring in the active – inactive interconversion and provide tools for real‐time identification and quantitation of the two protein forms in solution.
Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by ...temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady‐state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high‐throughput drug screening campaigns.
In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon
(Sa-MVs). The most abundant proteins in Sa-MVs are ...the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10-11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from
(LUV
), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUV
) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUV
, and LUV
were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan's GP and red-edge excitation shift (REES) values in Sa-MVs and LUV
decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan's REES values are high (9.3-18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUV
and LUV
) and low (0.4-5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan's excited state dipole moment, and "solvent" reorientation around Laurdan's chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan's lifetime.
Evidence and arguments are summarized that suggest that intrinsic (tryptophan) protein fluorescence provides an excellent and convenient signal for monitoring both GEF (guanine nucleotide exchange ...factor) and GAP (GTPase activating protein) activity of a large number of small GTPases. In addition, post-translational modifications of Rab proteins occurring in a region known to be a hot spot for such modifications also lead to fluorescence changes that can be accurately monitored in a time-dependent manner. It is suggested that intrinsic fluorescence should be the first method chosen for monitoring such reactions of tryptophan-containing small GTPases.
•Intrinsic protein fluorescence - a quick and powerful tool to monitor GEF/GAP activity and post-translational modifications.•Evidence suggests physiological and also pathological importance of post-translational modifications in the switch II region.•Intrinsic fluorescence should be the first method chosen for monitoring such reactions of tryptophan-containing small GTPases.
•Increased level of LPE induced a rise in the phase transition temperature of lipids.•Increased content of LPE was accompanied by increased thermostability of porin.•The thermostabilizing effect of ...lipids is due to their effect on tertiary structure of porin.•Conformational changes may impede porin function under stress in bacteria.
The present work aimed to compare the effects of different lysophosphatidylethanolamine (LPE) content in lipids derived from Yersinia pseudotuberculosis cells exposed and not exposed to phenol on the conformation of OmpF-like porin of these bacteria. Differential scanning calorimetry and intrinsic protein fluorescence showed that the 2.5-fold increase of LPE content and the corresponding increase in the phase transition temperature of bacterial lipids were accompanied by enhanced protein thermostability. Integral conformational rearrangement of protein was supported by drastic changes in the microenvironment of the tryptophan residues, likely resulting in a convergence of monomers in trimeric porin and exposure of outer tryptophan residues to the water environment. These conformational changes may impede the porin channel permeability under stress conditions in bacteria.
The number of binding sites for a given solute in a protein is a most relevant parameter. This number can be derived from fluorescence quenching data which provides the fraction of sites occupied at ...a given free solute concentration. Data are generally treated according to Scatchard´s or Ward´s equations. Lately, a double logarithmic plot of the data has been extensively used with this purpose. The present communication discus the validity of this procedure. It is concluded that this type of plot provides an evaluation of the stoichiometry (molecularity) of the binding process but not the number of equivalent binding sites per protein.
Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender ...β-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded β-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 β-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the β-helix is small. It is located near the C-terminal end of the β-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the β-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete β-helix.
Lysophosphatidyletnolamine (LPE) is one of enigmatic lipids of bacteria. It is generated from major membrane lipid - phosphatidylethanolamine at severe changes of the bacterial growth conditions. ...Accumulation of this phospholipid in cells of Gram-negative enterobacterium Yersinia pseudotuberculosis results in the enhanced thermostability of OmpF-like porin (YOmpF) from the same bacteria. The respective integral conformational rearrangements may disturb the channel permeability of protein under stress conditions. However, role of fatty acid composition of LPE in this effect remained unclear. Present work demonstrated that the level of unsaturated LPE is 3.5 times higher than saturated one in total LPE of bacterial cells exposed to stress (phenol treatment). Unsaturated 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (MOPE) and saturated LPE 1-palmitoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine (MPPE) oppositely affect the conformation of YOmpF. MOPE increases the protein thermal stability due to more dense packing of monomers in porin and preserves its trimeric form at elevated temperature, while MPPE weakens the contact between monomers and promotes dissociation of the protein.
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