Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.

Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom) or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Seccoo- motif that had 0.5% of the activity of the wild-type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between the enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as a donor of an electron to the substrate.

Photochromic copolymers containing 3-indolylfulgides/indolylfulgimides: synthesis and photochemical properties in toluene and as films.

Photochromic indolylfulgimides covalently attached to polymers have beneficial properties for optical switching. A 3-indolylfulgide and two 3-indolylfulgimides with one or two polymerizable styrene groups attached on the nitrogen atom(s) were synthesized. Copolymerization with methyl methacrylate (MMA) provided linear copolymers (one styrene group) or a cross-linked copolymer (two styrene groups). The properties of the monomers and copolymers in toluene or as thin films were characterized. The new copolymers were photochromic (reversible Z-to-C isomerization), absorbed visible light, and revealed good thermal and photochemical stability. At room temperature, all copolymer films showed no loss of absorbance after 5 weeks. At 80 °C in either toluene or as films, the Z-forms copolymers were less stable than the C-form copolymers, which showed little or no degradation after 400 h. The degradation rate due to repeated ring-closing - ring opening cycles was less than 3% per 100 cycles. The cross-linked copolymer showed photochemical stability comparable to monomeric fulgides in toluene, <1% per 100 cycles. In general, the properties of the linear and cross-linked copolymers were similar to the corresponding monomers in toluene. In films, the conformations of the Z-form were restricted due to the matrix indicating that the preparation of films from the C-form is advantageous.

Oxidative folding of lysozyme with aromatic dithiols, and aliphatic and aromatic monothiols.

In vitro protein folding of disulfide containing proteins is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. In this study, we examined redox buffers containing asymmetric dithiols 1-5, which possess an aromatic and aliphatic thiol, and symmetric dithiols 6 and 7, which possess two aromatic thiols, for their ability to fold reduced lysozyme at pH 7.0 and 8.0. Most in vivo protein folding catalysts are dithiols. When compared to glutathione and glutathione disulfide, the standard redox buffer, dithiols 1-5 improved the protein folding rates but not the yields. However, dithiols 6 and 7, and the corresponding monothiol 8 increased the folding rates 8-17 times and improved the yields 15-42% at 1mg/mL lysozyme. Moreover, aromatic dithiol 6 increased the in vitro folding yield as compared to the corresponding aromatic monothiol 8. Therefore, aromatic dithiols should be useful for protein folding, especially at high protein concentrations.

Photochromic properties of a water-soluble methyl carboxylic acid indolylfulgimide.

Photochromic fulgides and fulgimides have been identified as promising materials for applications in optical memory media, optical switches, and sensors. For applications in humid environments or biological systems, hydrolytic stability is crucial. A new photochromic methyl carboxylic acid indolylfulgimide was synthesized to improve hydrolytic stability in aqueous solution. The UV-vis spectra, extinction coefficient, thermal stability, and photochemical stability of the fulgimide were characterized in 50 mM sodium phosphate buffer (pH 7.4). The open and closed forms were both stable in buffer. At 37 °C after 500 h, the open forms of the fulgimide showed no degradation within experimental error (1-2%) by (1)H NMR and 2.3% decomposition by UV-vis spectroscopy. The closed form degraded 22% and 11% after 500 h at 37 °C in buffer by UV-vis and (1)H NMR data, respectively. In addition, the fulgimide cycled back and forth between the open and closed forms 80 times before degrading by 20% in buffer. The methyl group at the bridging position of the fulgimide significantly increased the thermal stability by overcoming the rapid hydrolysis of the trifluoromethyl group.

Increasing the efficiency of the ring-opening reaction of photochromic indolylfulgides by optical pre-excitation.

For three indolylfulgides the quantum efficiency of the ring-opening reaction upon pre-excitation is investigated in a multipulse experiment. The quantum efficiency grows by factor of up to 3.4, when the pre-excitation pulse immediately precedes the excitation process. The change in quantum efficiency after pre-excitation is discussed as a function of reaction time, steady-state quantum efficiency and energetic barriers in the excited electronic state. The observed differences can be explained by the molecular properties of the investigated indolylfulgides.

Synthesis and optical properties of aqueous soluble indolylfulgimides.

Three novel aqueous soluble fulgimides, trifluoromethyl carboxylic acid indolylfulgimide 4, dicarboxylic acid indolylfulgimide 5, and carboxylic acid indolylfulgimide 6, were synthesized. Both 4 and 5 can switch back and forth between open and closed forms upon illumination with specific wavelengths of light, whereas 6 can only switch from the closed form to the open form. In sodium phosphate buffer (pH 7.4) at 37 degrees C, an unusual hydrolysis of the trifluoromethyl group of the closed form of 4 resulted in 5, which has an additional carboxylic acid group. The closed form of 5 was further decarboxylated to generate 6, which was not photochromic. In buffer, the open form of 4 degraded 20% after 10 days, while the closed form of 4 was converted to 5 rapidly. In buffer, both forms of 5 degraded less than 20% after 21 days at 37 degrees C, and 5 underwent 670 photochemical cycles before degrading by 20%. It is the most robust fulgimide yet reported in aqueous solution.

Ring-opening reaction of a trifluorinated indolylfulgide: mode-specific photochemistry after pre-excitation.

The ring-opening reaction of a trifluorinated indolylfulgide has been studied as a function of temperature and optical pre-excitation where it was found that reaction times decreased as temperature increased from 10.3 ps at 12 degrees C to 7.6 ps at 60 degrees C. Simultaneously, the quantum yields for the ring-opening reaction grew from 3.1% (12 degrees C) to 5.0% (60 degrees C). When the reaction was started from a non-equilibrium state generated by a directly preceding ring-closure process, the ring-opening reaction became faster and the quantum efficiency increased by more than a factor of three. Analysis of the experimental results points to mode-specific photochemistry in that the promoting, photochemically active modes of the photoreaction are efficiently excited by the directly preceding ring-closure reaction.

Comparison of the oxidative folding of lysozyme at a high protein concentration using aromatic thiols versus glutathione.

The production of proteins using recombinant DNA technology often requires the use of in vitro protein folding. In order to facilitate in vitro protein folding, a redox buffer is added to the protein folding mixture. The redox buffer is composed of a small molecule disulfide and/or a small molecule thiol. Recently, redox buffers containing aromatic thiols have been shown to be an improvement over traditional redox buffers such as glutathione. For in vitro protein folding to be relevant to protein production on a larger scale, high protein concentrations are required to avoid large volumes of folding buffer. Therefore, we investigated the in vitro folding of lysozyme at 1 mg/mL instead of the traditional 0.1 mg/mL. Aromatic thiols and aromatic disulfides were compared directly with glutathione and glutathione disulfide, the most commonly used redox buffer. Folding experiments at pH 7 using aromatic thiols increased the yield by 20-40% and the folding rate constants by as much as 11 times relative to glutathione. At pH 8, improvements in yields of up to 25% and up to a 7-fold increase in folding rate constants were demonstrated. The effect of aromatic disulfide concentration was also investigated.

Wavelength and solvent independent photochemistry: the electrocyclic ring-closure of indolylfulgides.

A wavelength and solvent dependent study of a photochromic indolylfulgide is presented. The ring-closure reaction is characterized using stationary and time-resolved spectroscopy with femtosecond time resolution. After excitation into the first excited singlet state (S(1)) the photoprocesses proceed on ultrafast timescales (0.3-0.45 ps) in both polar and non-polar solvents. Excitation into higher electronic states results in similar reaction kinetics as found for S(1) excitation. A simple kinetic scheme can be established for the photoprocesses under all different experimental conditions: as expected from organic textbooks neither the solvent surroundings nor the excitation wavelength strongly alter the reaction scheme. The experimental study demonstrates that the ring-closure reaction of photochromic indolylfulgides can be considered as a very robust photoprocess: this fact may lead to a great variety of different applications where the reaction dynamics of the molecular switch are not disturbed by any surrounding effects.

Stability and reaction dynamics of trifluorinated indolylfulgides.

Quantum efficiencies and ultrafast dynamics of the ring-closure and ring-opening reaction of a trifluorinated dicyclopropyl indolylfulgide with improved photostability are investigated by stationary and ultrafast absorption spectroscopy. The ring-closure reaction occurs on the time scale of 200 fs and is found to be temperature independent (T = 287 - 333 K). However, an activated behaviour is observed for the ring-opening reaction. A comparison with the corresponding non-substituted indolylfulgide shows that the dicyclopropyl group favours the open isomer via lower cyclisation and higher cycloreversion quantum efficiencies and faster dynamics of the ring-opening reaction.

Accelerated and efficient photochemistry from higher excited electronic states in fulgide molecules.

The photoinduced electrocyclic ring-opening of a fluorinated indolylfulgide is investigated by stationary and ultrafast spectroscopy in the UV/vis spectral range. Photoreactions, initiated by optical excitation into the S(1) (570 nm) and S(N) (340 nm) absorption band of the closed isomer, lead to considerable differences in reaction dynamics and quantum yields. Transient absorption studies point to different reaction pathways depending on the specific excitation wavelength: excitation into the S(1) state leads to the known reaction behavior with a picosecond decay to the ground state and a small quantum yield of 7% for the photoproduct. The S(N) state shows an unexpected long lifetime of 0.5 ps. The photoreaction starting from the S(N) state leads to a large extent directly to the product ground state and back to the educt ground state. This results in an increased reaction quantum yield of 28%. In contradiction to Kasha's rule, the S(1) state is only populated with an efficiency of 38%. The observed behavior strongly differs from the expected picture with fast relaxation into the S(1) state and a subsequent ring-opening reaction starting from the lowest excited electronic state. Quantum chemical calculations confirm and complement the experimental findings allowing a sound molecular interpretation to be obtained.

Small-molecule catalysts of oxidative protein folding.

Oxidative protein folding occurs both in vivo and in vitro and involves the formation and rearrangement of protein disulfide bonds (SS bonds). In vivo these reactions are catalyzed by enzymes, including the eukaryotic enzyme protein disulfide isomerase (PDI). Using the physical properties of PDI as a guide, several small-molecule catalysts of oxidative protein folding have been designed, synthesized, and tested. These small molecules can improve the folding rate of the model substrate ribonuclease A by a factor of over 10 and improve the yield by up to a factor of 3 over traditional conditions. The molecules have also been demonstrated to significantly improve the in vivo folding of proteins as well.

Balancing conformational and oxidative kinetic traps during the folding of bovine pancreatic trypsin inhibitor (BPTI) with glutathione and glutathione disulfide.

The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulfide-containing proteins from their reduced form, as well as for protein folding in general. Many extracellular proteins and most pharmaceutically important proteins contain disulfide bonds. Under traditional conditions, 0.125 mM glutathione disulfide (GSSG) and no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a two disulfide intermediate), or a productive route via N' (and other two disulfide intermediates which are in rapid equilibrium with N'). Both routes have the rearrangement of disulfide bonds as their rate-determining steps. However, the effects of the composition of the redox buffer, GSSG and GSH, on folding has not been extensively investigated. Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG and 5 mM GSH than it does under traditional conditions. These conditions, which are similar to those found in vivo, result in a doubly mixed disulfide between N' and glutathione, which acts as an oxidative kinetic trap as it has no free thiols. However, with 5 mM GSSG and 5 mM GSH the formation of the double mixed disulfide is compensated for by N* being less kinetically stable and the more rapid conversion of the singly mixed disulfides between N' and glutathione to native protein (N). Thus a major rate-determining step becomes the direct conversion of a singly mixed disulfide to N, a growth-type pathway. Balancing the formation of N* and its stability versus the formation of the doubly mixed disulfide and its stability results in more efficient folding. Such balancing acts may prove to be general for other disulfide-containing proteins.

Thermal Stability and Photochromic Properties of a Fluorinated Indolylfulgimide in a Protic and Aprotic Solvent.

The photochromic properties and thermal stability of a newly synthesized fluorinated N-ethoxycarbonylmethyl indolylfulgimide substituted on the imide nitrogen were examined in a protic and aprotic environment. The absorption spectra and extinction coefficients of the Z- and C-forms of the fluorinated indolylfulgimide (open and closed, respectively) were measured in a binary 70/30 ethanol/water system and in toluene. The results demonstrated a favorable bathochromic shift of the absorption maxima for both the open and closed forms of the fulgimide when the solvent was changed from aprotic toluene to protic aqueous ethanol. In addition, the photochemical stability of the new fulgimide was found to be high (0.056% and 0.020% degradation each time the fulgimide is cycled between the open and closed form in 70/30 ethanol/water and in toluene, respectively). The thermal stability of both forms of the fulgimide in 70/30 ethanol/water at 50 degrees C, toluene at 80 degrees C, and polymer film (PMMA) at 80 degrees C was measured using UV-Vis and/or (1)H NMR spectroscopy. Both forms of indolylfulgimide display high hydrolytic stability in 70/30 ethanol/water at 50 degrees C, with the Z- and C-forms degrading 1.3%/day and 1.2%/day respectively based on (1)H NMR data. At 80 degrees C in toluene the less stable Z-form lost about 20%/day.

Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers.

Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important parameter for efficient in vitro folding is the composition of the redox buffer, a mixture of a small molecule thiol and small molecule disulfide. The effects of different redox buffers on protein folding, however, have received limited attention. The oxidative folding of denatured reduced lysozyme was followed in the presence of redox buffers containing varying concentrations of five different aromatic thiols or the traditional aliphatic thiol glutathione (GSH). Aromatic thiols eliminated the lag phase at low disulfide concentrations, increased the folding rate constant up to 11-fold, and improved the yield of active protein relative to GSH. The yield of active protein was similar for four of the five aromatic thiols and for glutathione at pH 7 as well as for glutathione at pH 8.2. At pH 6 the positively charged aromatic thiol provided a higher yield than the negatively charged thiols.

ortho- and meta-substituted aromatic thiols are efficient redox buffers that increase the folding rate of a disulfide-containing protein.

Thiol based redox buffers are used to enhance the folding rates of disulfide-containing proteins in vitro. Traditionally, small molecule aliphatic thiols such as glutathione are employed. Recently, we have demonstrated that aromatic thiols can further enhance protein-folding rates. In the presence of para-substituted aromatic thiols the folding rate of a disulfide-containing protein was increased by 4-23 times over that measured for glutathione. However, several important practical issues remain to be addressed. Aromatic thiols have never been tested in the presence of denaturants such as guanidine hydrochloride. Only two of the para-substituted aromatic thiols previously examined are commercially available. To expand the number of aromatic thiols for protein folding, several commercially available meta- and ortho-substituted aromatic thiols were studied. Furthermore, an ortho-substituted aromatic thiol, easily obtained from inexpensive starting materials, was investigated. Folding rates of scrambled ribonuclease A at pH 6.0, 7.0 and 7.7, with ortho- and meta-substituted aromatic thiols, were up to 10 times greater than those with glutathione. In the presence of the common denaturant guanidine hydrochloride (0.5M) aromatic thiols provided 100% yield of active protein while maintaining equivalent folding rates.

Increased catalytic activity of protein disulfide isomerase using aromatic thiol based redox buffers.

PDI is an enzyme that acts as a chaperone, shufflase, and oxidase during the folding of disulfide-containing proteins. The ability of aromatic thiols to increase the activity of PDI-catalyzed protein folding over that of the standard thiol glutathione (GSH) was measured. 4-Mercaptobenzoic acid (ArSH) increased the activity of PDI by a factor of three.

Effects of redox buffer properties on the folding of a disulfide-containing protein: dependence upon pH, thiol pKa, and thiol concentration.

Aliphatic thiols are effective as redox buffers for folding non-native disulfide-containing proteins into their native state at high pH values (8.0-8.5) but not at neutral pH values (6-7.5). In developing more efficient and flexible redox buffers, a series of aromatic thiols was analyzed for its ability to fold scrambled ribonuclease A (sRNase A). At equivalent pH values, the aromatic thiols folded sRNase A 10-23 times faster at pH 6.0, 7-12 times faster at pH 7.0, and 5-8 times faster at pH 7.7 than the standard aliphatic thiol glutathione. Similar correlations between thiol pK(a) values and folding rates at each pH value suggest that the apparent folding rate constants (k(app)) are a function of the redox buffer properties (pH, thiol pK(a) and [RSH]). Fitting the observed data to a three-variable model (logk(app)=-4.216(+/-0.030)+0.5816(+/-0.0036)pH-0.233(+/-0.004)pK(a)+log(1-e(-0.98(+/-0.02)[RSH]))) gave good statistics: r2=0.915, s=0.10.

Aromatic thiol pKa effects on the folding rate of a disulfide containing protein.

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.