Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes

In an attempt to develop a label-free electrochemical method for detection of changes in protein structures based on oxidizability of tyrosine and tryptophan residues we tested different types of carbon electrodes. We found that using edge plane pyrolytic graphite electrode (EPGE) we can discriminate between native and denatured forms of human serum albumin (HSA) and of other proteins, such as bovine and chicken serum albumin, aldolase and concanavalin. Treatment of natively unfolded α-synuclein with 8 M urea resulted only in a small change in the tyrosine oxidation peak, in a good agreement with absence of highly ordered structure in this protein. Using square wave voltammetry with EPGE we were able to follow the course of HSA denaturation at different urea concentrations. The electrochemical denaturation curve agreed reasonably well with that based on intrinsic fluorescence of tyrosine and tryptophan. It can be expected that the electrochemical method will be applicable to a large number of proteins and may become useful in biomedicine and proteomics.     

Reactivity of Singlet Oxygen Toward Proteins: The Effect of Structure in Basic Pancreatic Trypsin Inhibitor and in Ribonuclease A

The reactions of singlet oxygen, ¹O₂, with large peptides have been described previously. It was found that even in these systems, which in their native form are generally not supposed to possess a stable structure in solution, the polypeptide does impede the access of ¹O₂ to the amino acids that react readily with ¹O₂. Here we describe the ¹0₂ reaction with two proteins of well-defined structure. The quenching of ¹O₂ by bovine pancreatic trypsin inhibitor (BPTI) and by ribonuclease A (RNase A) was compared to that of a solution at the same concentration as those of its constituent amino acids that react readily with ¹O₂. The proteins were studied in their native form, when partly denatured by splitting their S-S bonds and when fully denatured. It was found that while in the native form the quenching rate constant was seven times lower in BPTI (2.2 vs 15.2 times 10⁷WM^-1 s^-1) and three times lower in RNase A (11.0 vs 32 times 10⁷M^-l s^-1) than in a mixture of its constituent amino acid residues, it increased upon denaturation reaching in the fully denatured state the value of the corresponding amino acid mixture. More striking is the effect of the protein structure when comparing the fraction of the encounters between ¹O₂ and protein, which cause damage to the protein, as reflected in the decrease of its biological activity. This decrease is assumed to be due to the chemical (oxidative) reactions of ¹O₂ in the protein. In the exceptionally stable BPTI the fraction of such encounters was 0.05 and in RNase A it was 0.2, whereas for the amino acid tryptophan in solution, 0.7 of the collisions with ¹O₂ led to a chemical reaction.     

FLASH PHOTOLYSIS OF RIBONUCLEASE A

Abstract— Flash photolysis spectra show that ultraviolet irradiation of RNase (Λ > 250 nm) at pH 11.5 generates the hydrated electron and a long-lived transient with absorption maxima at 390 nm and 410 nm, attributed to the phenoxyl type radical from tyrosyl residues. Comparison of the initial yields with flash photolysis spectra obtained from aqueous tyrosine and mixtures of the chromophoric amino acids indicates that 3–4 tyrosyl residues are photoionized in the primary act. This process is almost completely quenched at pH 1–9, even though the p -alanylphenoxyl radical is obtained with tyrosine over this pH range and the accompanying electron is observed at pH 7. The negative result is not altered by denaturation of RNase with 8 M urea or heating to 70°C, suggesting that a primary chain interaction is responsible for the suppression of tyrosyl residue photolysis. This mechanism is supported by flash photolysis spectra of small peptides, showing that the initial radical yield from tyrosylglycylglycine is strongly quenched compared to tyrosine when the phenolic group is protonated. Comparion of this work with published results on fluorescence and inactivation quantum yields indicates that photochemical electron ejection from RNase in alkaline solutions takes place in the dissociable residues and does not contribute to loss of enzymic activity.     

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.     

19F NMR studies of the native and denatured states of green fluorescent protein

Biosynthetic preparation and (19)F NMR experiments on uniformly 3-fluorotyrosine-labeled green fluorescent protein (GFP) are described. The (19)F NMR signals of all 10 fluorotyrosines are resolved in the protein spectrum with signals spread over 10 ppm. Each tyrosine in GFP was mutated in turn to phenylalanine. The spectra of the Tyr --> Phe mutants, in conjunction with relaxation data and results from (19)F photo-CIDNP (chemically induced dynamic nuclear polarization) experiments, yielded a full (19)F NMR assignment. Two (19)F-Tyr residues (Y92 and Y143) were found to yield pairs of signals originating from ring-flip conformers; these two residues must therefore be immobilized in the native structure and have (19)F nuclei in two magnetically distinct positions depending on the orientation of the aromatic ring. Photo-CIDNP experiments were undertaken to probe further the structure of the native and denatured states. The observed NMR signal enhancements were found to be consistent with calculations of the HOMO (highest occupied molecular orbital) accessibilities of the tyrosine residues. The photo-CIDNP spectrum of native GFP shows four peaks corresponding to the four tyrosine residues that have solvent-exposed HOMOs. In contrast, the photo-CIDNP spectra of various denatured states of GFP show only two peaks corresponding to the (19)F-labeled tyrosine side chains and the (19)F-labeled Y66 of the chromophore. These data suggest that the pH-denatured and GdnDCl-denatured states are similar in terms of the chemical environments of the tyrosine residues. Further analysis of the sign and amplitude of the photo-CIDNP effect, however, provided strong evidence that the denatured state at pH 2.9 has significantly different properties and appears to be heterogeneous, containing subensembles with significantly different rotational correlation times.     

Hydration of thermally denatured lysozyme studied by sorption calorimetry and differential scanning calorimetry

We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.     

Theoretical study of the cosolvent effect on the partial molar volume change of staphylococcal nuclease associated with pressure denaturation

We explain the molecular mechanism of the effect of urea and glycerol cosolvents on the partial molar volume (PMV) change associated with the pressure denaturation of staphylococcal nuclease (SNase) protein recently observed in experiments. Native and denatured conformations of SNase are produced by using molecular dynamics simulations in water, and the PMV is obtained from the integral equation theory of molecular liquids called 3D-RISM, which is based on statistical mechanics. The PMV of the native SNase in water predicted by 3D-RISM theory is in good agreement with experiment. The PMV changes associated with pressure denaturation in water and in water-urea and water-glycerol mixtures are qualitatively reproduced. By analyzing the results obtained, we found two interesting cosolvent effects on the PMV: (1) both urea and glycerol cosolvents increase the PMVs of both native and denatured SNase compared to those in water and (2) both urea and glycerol cosolvents increase the PMV of denatured SNase more than that of native SNase. We also showed that these two observations can be explained in terms of the thermal volume, which is related to the packing effect of solvent molecules.     

Fluorescence and quenching comparative studies of halophilic and bovine glutamate dehydrogenase

Fluorescence techniques have been used to study the structural characteristics of many proteins. The halophilic enzyme NADP-glutamate dehydrogenase from Haloferax mediterranei is found to be a hexameric enzyme composed of identical subunits. Fluorescence spectra of native and denatured halophilic and bovine glutamate dehydrogenase (h-GDH and b-GDH) have been analysed. Native h-GDH presents the maximum emission at 338 nm, whereas for b-GDH the maximum appears at 332 nm. The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum in both cases. The unfolding of h-GDH is a gradual process, which is accompanied by a loss in enzyme activity. Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out. The tryptophan residues in the protein are more exposed to the solvent in h-GDH than in b-GDH. The total amount of tryptophan residues is nearly the same for both enzymes.     

The effect of trimethylamine N-oxide on RNase a stability

The thermal stability of bovine pancreatic ribonuclease (RNase A) has been investigated in the presence of trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements at neutral and acid pH conditions. It is well known that compatible osmolytes such as TMAO are effective in stabilizing protein structure and counteracting the denaturing the effect of urea and guanidinium hydrochloride (GuHCl). Calorimetric results show that TMAO stabilizes RNase A at pH 7.0 and does not stabilize the protein at pH 4.0. RNase A thermal denaturation in the presence of TMAO is a reversible two-state N ⇆ D process. We also show that TMAO counteracts the urea and GuHCl denaturing effect at neutral pH, whereas the counteracting ability is lost at acid pH.     

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

Introduction Esterases(EC3.1.1.x)representadiversegroup ofhydrolasescatalyzingthecleavageandformationof esterbonds.Theyarewidelydistributedinanimals,plantsandmicroorganisms.Becauseoftheiractivities inbothaqueousandnonaqueoussolventsystems,ester aseshavebe…     

Ornithine Carbamoyltransferase Unfolding States in the Presence of Urea and Guanidine Hydrochloride

Ornithine carbamoyltransferase folding/unfolding is a complex and not completely understood process. Our experimental results suggest that ornithine carbamoyltransferase interacts in a completely different way with urea and guanidine hydrochloride. In fact, we noticed that, increasing concentration from 0.0 to 8.0 M of the two additives, the enzyme follows a simple one-step transition mechanism in the presence of guanidine hydrochloride, with two macroscopic states (the native and the denatured one) significantly populated, whereas in the presence of urea a lot of different protein states can be detected and analyzed. Circular dichroism and UV-visible spectroscopy reveal a similar mechanism of perturbation at high temperature, with opening of hydrophobic core and a significant loss in α-helix structure in the presence of guanidine hydrochloride that cannot be found in the presence of urea.     

Direct electrochemistry of cytochrome c on nanotextured diamond surface

Nanotextured diamond surfaces with geometrical properties close to protein dimensions were used for the realization of direct electron transfer of cytochrome c (cyt c) without any covalent bonding. The peroxidase activity of native and denatured cyt c was also investigated. Cyclic voltammograms of native cyt c show quasi-reversible electron transfer reactions, while no heme redox activity is detected for denatured cyt c. Unfolding (denaturation) of cyt c can be achieved in the presence of hydrogen peroxide. Partially or fully denatured cyt c showed higher peroxidase activity than native cyt c. This is because denatured cyt c loses its tertiary structure and hydrogen peroxide is easier to access the heme redox center. The apparent Michaelis–Menten constant K_m for native and denatured cyt c has been determined to be 0.23 mM and 0.08 mM.     

Dehydrated native biopolymers - a unique representative of glassy systems

DSC study of native and denatured biopolymers with different chemical and steric structure was carried out in a wide range of temperatures and water contents. It was shown that all the native and denatured humid biopolymers studied are glassy systems. The residues of native structures surviving after partial dehydration prevent the glass transition at the glass transition temperatures of the denatured biopolymers. In dehydrated native biopolymers the processes of melting and glass transition take place in the same temperature range that leads to a large change of the heat capacity across denaturation.     

ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET-INDUCED INACTIVATION OF β-TRYPSIN*

Abstract— Stern-Volmer quenching constants for β-trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern-Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β-trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β-trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength.
Investigations of the ultraviolet-induced inactivation of β-trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β-trypsin is not involved in enzyme inactivation or tryptophan destruction.     

Denaturation of a Recombinant Cutinase from Fusarium solani in AOT-iso-Octane Reverse Micelles: a Steady-State Fluorescence Study

Abstract— Near UV absorbance and fluorescence spectroscopy show conformational changes of a recombinant cutinase from Fusarium solani incorporated in sodium-di-2-ethylhexyl sulfosuccinate (AOT)-iso-octane reversed micelles with W₀= [H₂O]/[AOT] = 20. Excitation spectra were used to decompose cutinase absorbance in its Trp and Tyr components, showing that the latter absorb red-shifted in the native cutinase in aqueous solution as compared to free Tyr, whereas in reverse micelles and denatured cutinase no shift is detected. Emission maxima variations (λ_max 303, 311 and 335 nm, respectively in aqueous, reverse micelles and thermally denatured cutinase) reflect progressive changes in the micropolarity of the environment and exposure of Trp residues at the protein surface. The encapsulation of cutinase in AOT-iso-octane reversed micelles induces a time-dependent denaturation measured by fluorescence intensity changes at 330 nm, which match the profile of enzyme activity loss in this media.     

还原变性核糖核酸酶在疏水性液-固界面上的复性

采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。     

Interaction of a Rhodococcus sp. trehalose lipid biosurfactant with model proteins: thermodynamic and structural changes

One major application of surfactants is to prevent aggregation during various processes of protein manipulation. In this work, a bacterial trehalose lipid (TL) with biosurfactant activity, secreted by Rhodococcus sp., has been identified and purified. The interactions of this glycolipid with selected model proteins have been studied by using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Bovine serum albumin (BSA) and cytochrome c (Cyt-c) have been chosen because of their quite different secondary structures: BSA contains essentially no β-sheets and an average 66% α-helix, whereas Cyt-c possesses up to 25% β-sheets and up to 45% α-helical structure. Differential scanning calorimetry shows that addition of TL to BSA at concentrations below the critical micelle concentration (cmc) shifts the thermal unfolding temperature to higher values. FTIR indicates that TL does not alter the secondary structure of native BSA, but the presence of TL protects the protein toward thermal denaturation, mainly by avoiding formation of β-aggregates. Studies on the intrinsic Trp fluorescence of BSA show that addition of TL to the native protein results in conformational changes. BSA unfolding upon thermal denaturation in the absence of TL makes the Trp residues less accessible to the quencher, as shown by a decrease in the value of Stern-Volmer dynamic quenching constant, whereas denaturation in the presence of the biosurfactant prevents unfolding, in agreement with FTIR results. In the case of Cyt-c, interaction with TL gives rise to a new thermal denaturation transition, as observed by DSC, at temperatures below that of the native protein, therefore facilitating thermal unfolding. Binding of TL to native BSA and Cyt-c, as determined by ITC, suggests a rather nonspecific interaction of the biosurfactant with both proteins. FTIR indicates that TL slightly modifies the secondary structure of native Cyt-c, but protein denaturation in the presence of TL results in a higher proportion of β-aggregates than in its absence (20% vs 3.9%). The study of Trp fluorescence upon TL addition to Cyt-c results in a completely opposite scenario to that described above for BSA. In this case, addition of TL considerably increases the value of the dynamic quenching constant, both in native and denatured protein; that is, the interaction with the glycolipid induces conformational changes which facilitate the exposure of Trp residues to the quencher. Considering the structures of both proteins, it could be derived that the characteristics of TL interactions, either promoting or avoiding thermal unfolding, are highly dependent on the protein secondary structure. Our results also suggest the rather unspecific nature of these interactions. These might well involve protein hydrophobic domains which, being buried into the protein native structures, become exposed upon thermal unfolding.     

REACTIVITY OF SINGLET OXYGEN TOWARD LARGE PEPTIDES

Abstract— The reactions of singlet oxygen, ¹O₂, with amino acids and their derivatives have been studied previously. It was found that only five amino acid residues interact readily with ¹O₂. Here we describe its reactions with the large peptides melittin, neuropeptide Y (NPY) and insulin in their native and in their denatured forms. The singlet oxygen quenching by a polypeptide was compared with that of a solution at the same concentration as those of its constituent amino acids, which are known to react efficiently with ¹O₂. It was found that the quenching rate by such a mixture exceeded that of the polypeptides in their native form. The ratio of the rate constants for NPY to that of the corresponding amino acid mixture in solution was 0.75. For melittin in its monomeric form it was 0.83 and for a tetramer of melittin (at high ionic strength) it was 0.70. For native insulin the ratio of the rate constants was 0.55. For oxidized insulin with its -S-S- bridges opened the figure became 0.80. However, the quenching by all the polypeptides in their fully denatured form (in the presence of 6 M urea) equalled that of the corresponding amino acid mixtures. Although polypeptides are generally supposed not to possess a stable secondary structure in solution the effects are explained by shielding of some of the reactive amino acid residues in the chain by temporary folding or incipient secondary structures of the native polypeptide.
It is shown that the kinetics for a homogeneous solution of quenchers applies also to measurements in a polypeptide solution where the quenchers are localized along the polypeptide backbone and thus form clusters in solution.     

脲或盐酸胍溶液中变性溶菌酶复性过程中集聚体的研究

建立在蛋白质变性-复性三态模型的基础上, 给出了一个描述在变性液中变性蛋白质复性时蛋白质浓度和其复性率的关系式. 通过这个关系式, 可以获得两个重要的描述蛋白质变性-复性体系特征的参数, 一个是包含在一个集聚体分子中的变性蛋白质的分子数目n, 另一个是蛋白质从原始态到形成集聚体过程中的表观集聚平衡常数K. 以三种溶菌酶在脲和盐酸胍溶液中的变性-复性过程对此方程进行了验证, 结果表明所给出的方程能够很好地描述三种溶菌酶在这两种变性液中的复性结果, 三种溶菌酶在两种变性液中有形成二分子集聚体的趋势. 变性溶菌酶在复性过程中的电泳和高效凝胶排阻色谱也同时能够监测到复性过程中集聚体的形成, 并且监测结果与上述方程所得的结果一致.     

Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin

Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.