Watson J. Lees

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.

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 Kashas 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.

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.

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.

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.

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.

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.

Going Non-native To Improve Oxidative Protein Folding

The efficient folding of disulfide-containing proteins has been a continuing challenge for researchers and for the biotechnology industry, as many proteins of pharmaceutical interest contain disulfide bonds. During oxidative protein folding, cysteine thiols ( SH) are oxidized to the native set of disulfide bonds ( SS ), through three different types of reaction: the oxidation of protein thiols to native or non-native protein disulfides, the reduction of protein disulfides back to protein thiols, and the rearrangement of disulfide bonds within the protein. Given the complexity of the process, especially for disulfide-rich proteins, it is not surprising that the rate and yield of oxidative protein folding can be limited by the formation of kinetically trapped intermediates. The folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been studied in detail by Creighton and Kim and contains two kinetically trapped intermediates. Native BPTI contains 58 amino acids and three disulfide bonds between cysteines 30 and 51, 14 and 38, and 5 and 55, of which two (30–51 and 5–55) are buried and one (14–38) is solvent exposed. Weissman and Kim demonstrated that during oxidative protein folding only intermediates with native disulfide bonds accumulate (Scheme 1, black boxes). The disulfide bonds within an intermediate are indicated within the boxes. Both N’ [30–51, 14–38] and N* [5–55, 14–38], which contain two native disulfide bonds, are kinetically stable as the two remaining free thiols are buried inside the protein and only react slowly with one of the other previously formed disulfide bonds in BPTI and/or an external disulfide. That is not to say that intermediates with non-native disulfide bonds do not occur along the folding pathway or that they are not important, a topic of some controversy, but they do not accumulate and act as kinetic traps that slow down the folding process. 6, 7] The oxidative folding of BPTI in vitro is very slow due to the formation of the two kinetic traps. At pH 7.3, it can take weeks to fold reduced protein to native protein in the traditional folding buffer, which contains glutathione and glutathione disulfide (GSSG). 9] With the seleno analogue of glutathione disulfide, glutathione diselenide (GSeSeG), the folding time was re-

Photoreaction from a light generated non-equilibrium state

We report on the acceleration of the S1 photoreaction combined with the dramatic increase of the photochemical quantum efficiency, when the reaction is directly preceded by another ultrafast photoreaction.