Study of the interactions between lysozyme and a fully-fluorinated surfactant in aqueous solution at different surfactant-protein ratios

The interactions of a fluorinated surfactant, sodium perfluorooctanoate, with lysozyme, have been investigated by a combination of UV absorbance, electrical conductivity and dynamic light scattering to detect and to characterize the conformational transitions of lysozyme. By using difference spectroscopy, the transition was followed as a function of surfactant concentration, and the data were analyzed to obtain the Gibbs energy of the transition in water (DeltaGw(o)) and in a hydrophobic environment (DeltaGh(o)) for saturated protein-surfactant complexes. Electrical conductivity was used to determine the critical micelle concentration of the surfactant in the presence of different lysozyme concentration. From these results, the average number of surfactant monomer per protein molecule was calculated. Finally, dynamic light scattering show that only changes in the secondary structure of the protein can be observed.     

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.     

Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering

To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein; disordered structures add as the pD decreases. Moreover, beta-aggregates seem to contribute only to oligomers formation, as showed by the good correlation between kinetics of scattering intensity and IR absorption intensity. These results indicate for BSA a general mechanism of aggregation composed by partial unfolding of the tertiary structure and by the decrease of alpha-helix and random coil contents in favor of beta-sheet aggregates. This mechanism strictly depends on pD and gives rise to almost two distinct types of macromolecular aggregates.     

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.     

The mechanism for thermal-enhanced chaperone-like activity of α-crystallin against UV irradiation-induced aggregation of γD-crystallin

Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.     

Multispectroscopic studies on the molecular interactions between bovine γ-globulin and borohydride-capped silver nanoparticles

Interactions between bovine γ-globulin (BGG) and borohydride-capped silver nanoparticles (BAgNPs) were studied using dynamic light scattering (DLS) and spectroscopic techniques such as UV–vis spectroscopy, fluorescence, and circular dichroism. The results were compared with earlier reported interactions between γ-globulin and citrate-coated AgNPs (CAgNPs). BAgNPs were synthesized and characterized. Irrespective of the coating on AgNPs, nanoparticles had formed ground-state complexes with the protein. CAgNPs, as well as BAgNPs had caused static quenching of tryptophan (Trp) fluorescence of the protein. The change in the capping agent from citrate to borohydride weakened the binding of nanoparticles with the protein. But the same change in capping agent had increased the fluorescence quenching efficiency of AgNPs. Hydrogen bonding and van der Waals interactions were involved in BGG–BAgNPs complex similar to the CAgNPs complex with γ-globulin. Polarity of the Trp microenvironment in BGG was not altered using BAgNPs as opposed to CAgNPs, as supported using synchronous and three-dimensional fluorescence. Resonance light scattering experiments also suggested nano-bio conjugation. Far-UV and near-UV circular dichroism (CD) spectra respectively pointed towards changes in the secondary and tertiary structure of BGG by BAgNPs, which was not observed for CAgNPs.     

The effect of ionic strength, temperature, and pressure on the interaction potential of dense protein solutions: from nonlinear pressure response to protein crystallization

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.     

Effect of pressure on the tertiary structure and dynamics of folded basic pancreatic trypsin inhibitor

The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein.     

Peptide-protein complex from cattle sclera: Structural aspects and chaperone activity

The influence of temperature and chaotropic agents on the spatial organization of the peptide-protein complex isolated from cattle sclera at the level of secondary structure was studied by UV, CD spectroscopy, and dynamic light scattering. It is shown that this complex has high conformational thermostability. The point of conformational thermal transition (65 °C) was determined, after which the peptide-protein complex passes into a denatured stable state. It was found that the peptide-protein complex in aqueous solutions forms thermostable nanosized particles. It was shown that the peptide-protein complex isolated from cattle sclera shows the properties of chaperone, an inhibitor of model protein aggregation induced by dithiothreitol.     

Chaperone Potential of Pulicaria undulata Extract in Preventing Aggregation of Stressed Proteins

This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.     

Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90-232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90-232 showed an infrared absorbance spectrum typical of proteins with a predominant alpha-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 m guanidine hydrochloride), the secondary structure of SHaPrP90-232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel beta-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in alpha-helical structures and the increase in beta-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No beta-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90-232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90-232. A new model for the structural transition and aggregation process of the prion protein is described.     

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.     

Quaternary conformational stability: The effect of reversible self‐association on the fibrillation of two insulin analogs

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far‐UV CD) and second derivative Fourier transform infrared (2D‐FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α‐helical in native insulin to predominantly β‐sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self‐associate. Composition gradient multi‐angle light scattering (CG‐MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant‐induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first‐order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359–2370. © 2011 Wiley Periodicals, Inc.     

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.     

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8 M urea (pH 8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH 5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.     

Conformational lability of two molecular chaperones Hsc70 and gp96: effects of pH and temperature

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.     

Effect of environmental conditions on aggregation and fibril formation of barstar

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross- structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-statefibril type Ifibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.Abbreviations CD circular dichroism - DLS dynamic light scattering - EM electron microscopy - SLS static light scattering - SAXS small-angle X-ray scattering - SOXS solution X-ray scattering     

Thermo- and photoinduced aggregation of alpha-crystallin

Some properties of bovine alpha-crystallin, in particular its thermo- and photoaggregation, were studied by fluorescent spectroscopy of tryptophan residues and the probe 8-anilino-1-naphthalenesulfonate and light scattering. The effective diameter of a globule of native alpha-crystallin was 90 A, as estimated from the data on the polarization and lifetime of 8-anilino-1-naphthalenesulfonate using the Levshin-Perren equation, and increases at an aggregation of no less than 140 A. The increase in the intensity of tryptophan fluorescence of alpha-crystallin during its thermo- and photodenaturation with the formation of aggregates is due to local conformational changes in the surroundings of tryptophan residues and light scattering. Tryptophan residues are buried in the interior of the aggregates. The thermoaggregation of the protein occurs not only at high temperatures. By approximating the experimental time dependence of slow spontaneous aggregation to the range of large times, the time of denaturation aggregation t(e) was found. For alpha-crystallin (at a concentration of 0.8 mg/ml in phosphate buffer at pH 8.4), t(e) at 70 degrees C is 100 h. This approach can be used in finding t(e) for any protein during its thermal treatment or long-term storage.     

Amyloid fibrils formation and amorphous aggregation in concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.     

Alternative prion structural changes revealed by high pressure

At high temperature, recombinant hamster prion protein (SHaPrP(90-231)) undergoes aggregation and changes from a predominantly alpha-helical to beta-sheet conformation. We then applied high pressure (200 MPa) to the beta-sheet-rich conformation. The aggregation was reversed, and the original tertiary and secondary structures were recovered at ambient pressure, after pressure release. The application of a pressure of 200 MPa thus allowed studying the heat-induced equilibrium refolding in the absence of protein aggregation. Prion protein unfolding as a function of high pressure was also investigated. Simple two-state, reversible unfolding transitions were observed, as monitored by spectral changes in the UV and fluorescence of the hydrophobic probe 8-anilino-1-naphthalene sulfonate. However, these heat- and pressure-induced conformers differed in their unfolding free energy. At pressures over 400 MPa, strong thioflavin-T binding was observed, suggesting a further structural change to a metastable oligomeric structure.