Transition Of Bovine Serum Albumin Research Articles

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

Suppression of Sunscreen Leakage in Water by Amyloid-like Protein Aggregates.

A sunscreen offers indispensable skin protection against UV damage and related skin diseases. However, due to the poor interfacial stability of sunscreen coatings on the skin, the synthetic ingredients in sunscreen creams easily fall off and enter aquatic environments, causing large ecological hazards and skin protection failure. Herein, we tackle this issue by introducing amyloid-like protein aggregates into a sunscreen to noticeably enhance the interfacial robustness of sunscreen coatings on the skin. The synthesis of such an agent to suppress sunscreen leakage can be achieved by manipulating the phase transition of bovine serum albumin (BSA) in a mild aqueous solution at room temperature. The resulting phase-transitioned BSA (PTB) aggregates effectively entrap the sunscreen ingredients to generate a uniform cream coating on the skin with robust amyloid-mediated interfacial adhesion stability. With continuous flushing in aquatic environments, such as salt water and seawater, this PTB-modified sunscreen (PTB sunscreen) coated on the skin maintains a retention ratio as high as >92%, which is 2-10 times higher than those of commercially available sunscreen products. The high retention ratio of the PTB sunscreen in aquatic environments demonstrates the great potential of amyloid-like protein aggregates in the development of leakage-free sunscreens with low ecosystem hazards and long-lasting UV protection in aquatic environments.

Following Structural Changes by Thermal Denaturation Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature (T ∼ 23-70 °C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H]12+ to [M + 16H]16+ at ∼23 °C) and narrow mobility distribution toward an unfolded-like, broad charge state (up to [M + 46H]46+ at ∼70 °C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature Tm ∼ 56 ± 1 °C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 μM), this method can detect nonspecific BSA dimers and trimers which dissociate early (Td ∼ 34 ± 1 °C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).

Determining protein structure with tyrosine bioconjugation

A hallmark of many neurodegenerative diseases like Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Lobar Dementia (FTLD) is the formation of misfolded proteins into aggregates in the neuronal cells. These proteins often have intrinsically disordered regions which are enriched in a low complexity of amino acids such as Serine, Glycine, Glutamic acid and Tyrosine. Disordered proteins are challenging to study with common techniques such as x‐ray crystallography and NMR. Furthermore, common biorthogonal chemical reactions which mostly target lysine and cysteine residues cannot be used to determine the protein fold due to the low variety of amino acid composition in these proteins. However, a unique characteristic of several disordered proteins involved in neurodegenerative disease is their enrichment for tyrosines. Here we have developed an approach to assess tyrosine accessibility as a measure of levels in native vs misfolded forms of the proteins.To measure tyrosine accessibility we employed the ability of 4‐Phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) to conjugate to tyrosine side‐chains. We assessed the targeted labeling of tyrosine’s for peptides, proteins, and cells. We have used this chemical approach to observe the transition of Bovine Serum Albumin (BSA), a well‐characterized and folded protein, to an unfolded state. We found that we could assess the local changes to protein structure by using LC‐MS/MS to quantify levels at each tyrosine position. We could confirm changes using fluorescent detection through click chemistry. Our long‐term goal is combining this approach and proteomics to quantify changes in cells to protein folding in comparable cellular states, such as disease and non‐diseased tissues.Support or Funding InformationNational Institute of Health, American Cancer Society

The effect of the stoichiometric ratio of zinc towards the fibrillation of Bovine Serum Albumin (BSA): A mechanistic insight

Fibrillation of proteins is a major cause of various neurodegenerative diseases and its exact mechanism of formation is yet unclear instead of extensive research. However, the role of metal ions influencing fibrillation of proteins is gaining more attention recently. Herein, we have investigated the role of various concentrations of the transition metal, Zn(II), on the fibrillation of Bovine Serum Albumin (BSA) at the physiological pH 7.4. Several biophysical and simulation techniques were employed in order to analyze the same. Thioflavin T intensity and residual protein investigations revealed that fibrillation of BSA was significantly decelerated and accelerated at 1:3 and 1:4 ratios of BSA-Zn(II), respectively; while it was found to be independent at other ratios (1:1 and 1:2). Fourier transform infrared spectroscopy analysis revealed that the transition of BSA from α-helical conformation to the β-sheet rich structure is greatly resisted at 1:3 ratio, however, the same is promoted at 1:4 ratio. Similarly, dynamic light scattering and field emission transmission electron microscopy analyses further confirmed the above observations. Furthermore, Isothermal Titration Calorimetry revealed the interaction of Zn(II) towards four binding sites of BSA with preferential affinities. Molecular dynamics studies predicted that at 1:3 ratio, the C- and N-terminal zones of BSA were least flexible owing to more stable conformation. Moreover, the solvent accessible surface area and structural analyses showed increase in hydrophilicity and more conserved secondary structure, respectively at 1:3 ratio. We propose that BSA fibrillation is indeed dependent on particular Zn(II) concentration, the temperature of the microenvironment of BSA, the number of binding sites exposed due to unfolding and the conformation after metal binding.

Secondary structure transitions of Bovine serum albumin induced by temperature variation

Secondary structure transitions of Bovine Serum Albumin (BSA) in H2O and D2O under temperature variation were investigated by in-situ IR spectral analysis and they were found to exhibit the same tendency with the only difference that the significant transition temperature of BSA's secondary structure moved towards a lower temperature in D2O. Before this significant transition temperature, the native secondary structures of BSA were essentially changing in a cooperative fashion with temperature variation, except for the intermolecular β-sheet (high-wavenumber) which increased as a result of thermal denaturation at a later stage. After the transition temperature, the relative contents of α-helix and extended chain continued to drop sharply and they were transformed into random coil, β-turn and β-sheet.

Tracking structural transitions of bovine serum albumin in surfactant solutions by fluorescence correlation spectroscopy and fluorescence lifetime analysis

The structural dynamics of proteins is crucial to their biological functions. A precise and convenient method to determine the structural changes of a protein is still urgently needed. Herein, we employ fluorescence correlation spectroscopy (FCS) to track the structural transition of bovine serum albumin (BSA) in low concentrated cationic (cetyltrimethylammonium chloride, CTAC), anionic (sodium dodecyl sulfate, SDS), and nonionic (pentaethylene glycol monododecyl ether, C12E5 and octaethylene glycol monododecyl ether, C12E8) surfactant solutions. BSA is labelled with the fluorescence dye called ATTO-488 (ATTO-BSA) to obtain steady fluorescence signals for measurements. We find that the diffusion coefficient of BSA decreases abruptly with the surfactant concentration in ionic surfactant solutions at concentrations below the critical micelle concentration (CMC), while it is constant in nonionic surfactant solutions. According to the Stokes-Sutherland-Einstein equation, the hydrodynamic radius of BSA in ionic surfactant solutions amounts to ∼6.5 nm, which is 1.7 times larger than in pure water or in nonionic surfactant solutions (3.9 nm). The interaction between BSA and ionic surfactant monomers is believed to cause the structural transition of BSA. We confirm this proposal by observing a sudden shift of the fluorescence lifetime of ATTO-BSA, from 2.3 ns to ∼3.0 ns, in ionic surfactant solutions at the concentration below CMC. No change in the fluorescence lifetime is detected in nonionic surfactant solutions. Moreover, by using FCS we are also able to identify whether the structural change of protein results from its self-aggregation or unfolding.

Photoacids as a new fluorescence tool for tracking structural transitions of proteins: following the concentration-induced transition of bovine serum albumin

Spectroscopy-based techniques for assessing structural transitions of proteins follow either an intramolecular chromophore, as in absorption-based circular dichroism (CD) or fluorescence-based tryptophan emission, or an intermolecular chromophore such as fluorescent probes. Here a new fluorescent probe method to probe the structural transition of proteins by photoacids is presented, which has a fundamentally different photo-physical origin to that of common fluorescent probes. Photoacids are molecules that release a proton upon photo-excitation. By following the steady-state and time-resolved emission of the protonated and de-protonated species of the photoacid we probe the environment of its binding site in bovine serum albumin (BSA) in a wide range of weight concentrations (0.001-8%). We found a unique concentration-induced structural transition of BSA at pH2 and at concentrations of >0.75%, which involves the exposure of its hydrophobic core to the solution. We confirm our results with the common tryptophan emission method, and show that the use of photoacids can result in a much more sensitive tool. We also show that common fluorescent probes and the CD methodologies have fundamental restrictions that limit their use in a concentration-dependent study. The use of photoacids is facile and requires only a fluorospectrometer (and preferably, but not mandatorily, a time-resolution emission system). The photoacid can be either non-covalently (as in this study) or covalently attached to the molecule, and can be readily employed to follow the local environment of numerous (bio-)systems.

Protein denaturation kinetic processes of a simple and a complex reaction mechanism analyzed by an iso-conversional method

The protein denaturation kinetic processes of a simple and a complex reaction mechanism represented by bovine serum albumin and hen egg-white lysozyme were analyzed by an iso-conversional method using differential scanning calorimetry. After differential scanning calorimetry using the iso-conversional method, the results were found to pose distinct contrasts between the two proteins. Bovine serum albumin showed an increasing peak temperature of the transition as the scan rate and protein concentration increased, whereas hen egg-white lysozyme exhibited almost constant peak temperature. The differential scanning calorimetry transition of bovine serum albumin was calorimetrically irreversible, while one part of hen egg-white lysozyme denaturation process was irreversible during which aggregation occurred and the other part was reversible. The iso-conversional method indicated that the value of bovine serum albumin apparent activation energy hardly varied with the degree of conversion, which showed that the denaturation kinetic process should conform to single reaction model. Using the master plots method, the most possible kinetic model for bovine serum albumin denaturation might be described by F n kinetic model. On the contrary, the hen egg-white lysozyme value of apparent activation energy decreased with the increase of degree of conversion. It was not a process involving the two standard reversible states, and can be described by the simple Lumry–Eyring model. The iso-conversional method provides new opportunities in exploring a simple and a complex reaction mechanism of protein denaturation.

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature-induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed-phase liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12-50 °C. The results showed that, in the temperature range 12-20 °C, BSA only existed in a single conformation state A, while in the temperature range 22-50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy-driven.

Thermodynamic and kinetic processes during the unfolding of BSA in the presence of the mycotoxin patulin

The effects of the mycotoxin patulin on the thermodynamics and kinetics of the transition of bovine serum albumin (BSA) in aqueous solution were studied by Differential Scanning Calorimetry and Photoluminescence methods. Results show that in the presence of patulin, the free enthalpy change during the transition of BSA was decreased by an average of ∼ 46 kJ/mol, the free energy change was decreased by ∼ 4 kJ/mol, and the activation energy fell from ∼ 1546 to ∼ 840 kJ/mol. These results indicate that the bioactivity of patulin is based on the kinetic rather than on the thermodynamic properties of the transition. This is the first evidence of the direct interaction of patulin with the free thiol-containing BSA, a process which could contribute to the adverse cyto- and genotoxic effects induced by patulin.

The environment controlled effect of thiacalix[4]arene on the transition thermodynamics and kinetics of bovine serum albumin

Complexation ability of water-soluble thiacalix[4]arene-tetrasulfonate towards three aromatic amino acids (Phenylalanine, Tyrosine and Tryptophane) was studied in water–ethanol mixtures by photoluminescence (PL) method as a function of the ethanol content of the bulk solutions. Job’s method followed by the application of the van’t Hoff theory was used to determine the thermodynamic parameters of the molecular association. Results show quite different thermodynamics of formation of calixarene–amino acid complexes at low and higher ethanol content of the solutions. The considerable stability of the individual calixarene–aromatic amino acids complexes supports their existence also in the case when the amino acids are in a protein. To test this idea the conversion rate, enthalpy and entropy change associated to the structural transition of BSA (bovine serum albumin) were investigated by Differential Scanning Calorimetry (DSC) in the absence and in the presence of calixarene. Results show that presence of calixarene changes significantly both the thermodynamics and the kinetics of the transition of BSA and the information collected for the individual calixarene–amino acid complexes gives insights about the possible processes at molecular level.

Influence of nanoparticle size on the pH-dependent structure of adsorbed proteins studied with quantitative localized surface plasmon spectroscopy

We have studied pH-dependent conformational transitions of Bovine Serum Albumin adsorbed onto different sizes of gold nanospheres. For larger spheres (D > 10 nm) there is evidence for a path-dependent extended state near pH 4, over a very small pH range. For smaller nanospheres (5 nm and 10 nm) the evidence for such a transition is either much weaker or completely suppressed. We suggest that the absence of the transition on small spheres is due to the fact that the protein adsorbed on such small spheres has already lost at least some of its tertiary structure. The results have important implications for the functionality of proteins adsorbed onto nanospheres or surfaces with nm scale roughness.

Influence of metal ions on folding pathway and conformational stability of bovine serum albumin

In order to understand the effects of metal ions on the structural stability of proteins, urea-induced unfolding transition of bovine serum albumin (BSA) in the presence of different kinds of metal ions (Zn 2+, Ca 2+, Cd 2+ and Co 2+) are characterized by far-UV circular dichroism (CD) and fluorescence spectra. Under neutral condition, there is a significant deviation of the unfolding transition curves of free protein by fluorescence and CD spectra. However, the unfolding curves of fluorescence and CD spectra turn to be coincident in the presence of Ca 2+ or Cd 2+ ions, indicating that the tertiary and secondary structures of BSA unfold simultaneously, i.e. a cooperative two-state behavior, while the deviation of them is enhanced in the presence of Zn 2+ or Co 2+ ions, suggesting that the unfolding remains a three-state behavior. Our results provide evidence that the metal ions alter the urea-induced unfolding pathway of BSA. Furthermore, the change of free energy (Δ G), the midpoint of chemical denaturation ( C m) and the solvent-exposed surface ( m) during the urea-induced unfolding process have also been obtained to characterize the effects of metal ions on the conformational stability of BSA.

Binding of odorants to individual proteins and their mixtures. Effects of protein denaturation and association. A plasticized globule state

The unfolding-association-refolding behaviour of proteins and their interactions with odorants are significant to the processing and flavoring of foods. Interactions of 2-octanone and vanillin (aliphatic and aromatic odorants) with individual proteins and their mixtures have been studied using: native, acid and thermally denatured bovine serum albumin (BSA), native and thermally denatured ovalbumin (OA) and bovine β-casein (BC). Equilibrium dialysis and differential scanning calorimetry methods were used to study flavor binding and flavor release in protein solutions. The binding of vanillin to native BSA exhibited two equivalent binding sites with binding constants in the order of 103 M−1 and these binding parameters were not markedly changed within the range of temperature from 25 to 63 °C. The binding site number was monotonously reduced to about zero as the pH was decreased from 6.4 to 2.5, consistent with the N⇔F transition of BSA. The complete loss of vanillin binding activity was also coupled with both acid and thermal denaturation of BSA. Neither the associated BC nor thermally denatured OA produced a detectable competition with BSA for the binding of vanillin or octanone. The associated BC did not bind vanillin. It has been proposed that the adsorption of hydrophobic low-molecular weight compounds acts to plasticize the globule state of the protein. Plasticized globular proteins seem to tend towards self-association and refolding with separation of hydrophobic ligands (e.g. odorants).

Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6–10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71°C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change Δ H for unfolding of 170 kcal/mol, the increase from 62 to 71°C corresponds to an additional Gibbs energy of stabilization (ΔΔ G) due to halothane of more than 4 kcal/mol. Analysis of the dependence of ΔΔ G on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.

PH-induced structural transitions of bovine serum albumin. Histidine pKa values and unfolding of the N-terminus during the N to F transition.

1. Titration curves have been constructed for all the resolvable resonances of defatted bovine serum albumin [pH meter readings in 2H2O solution (pH*) 2-10], including complete curves for six His residues and partial curves for a further four. Three His residues have unusually low pKa values (less than 5.5). 2. Abrupt discontinuities in the titration curves of nearly all resonances were observed near pH 4.3 (N to F transition), together with a marked loss of chemical shift dispersion at low pH*. No such loss of dispersion occurs in the high pH region previously associated with the N to B transition (approximately pH 8). The NMR data provide strong evidence that the N to F transition is not localised in domain III as had been previously suggested. 3. A localised unfolding of the N-terminal region occurs below pH* 4.5.

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation.

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.

The effect of the N-B transition of bovine and human serum albumins on the binding sites of fluorescent probes

The N(neutral)-B(alkaline) transition of bovine serum albumin (BSA) and human serum albumin (HSA) was investigated with three anionic and one cationic dansyl (dimethylamino-naphthalenesulfonyl, DNS) probes. The N-B transition of the HSA-probe complex was characterized by a large enhancement of the fluorescence intensity of the dansyl group, accompanied by a blue-shift of the maximum wavelength in the emission spectra, and large changes in the thermodynamic parameters of the binding process. Contrary to this, such changes were not observed for the BSA-probe complex. It is suggested that the conformational adaptability of HSA to the probes at the binding site increases in the B conformation, although the nature of the site on BSA is hardly affected by the transition.

Direct evidence for the involvement of domain III in the N-F transition of bovine serum albumin.

The domain III of bovine serum albumin containing residues 377-582 of the protein sequence was isolated and its behaviour in acid solution was studied. The fragment was found to undergo structural transformations over the pH range 3.5-4.5 known to cause N-F transition in serum albumin. On the other hand, an albumin fragment that was devoid of domain III was unable to exhibit such a transition. These results were consistent with a mechanism where N-F transition involves the separation of domain III from the rest of the albumin starts at about pH 4.3 and is completed at pH 3.5.