Characterizing Short-Lived Protein Folding Intermediates by Top-Down Hydrogen Exchange Mass Spectrometry

This work combines pulsed hydrogen/deuterium exchange (HDX) and top-down mass spectrometry for the structural characterization of short-lived protein folding intermediates. A custom-built flow device with three sequential mixing steps is used for (i) triggering protein folding, (ii) pulsed D(2)O labeling, and (iii) acid quenching. The earliest folding time point that can be studied with this system is 10 ms. The mixing device was coupled online to the electrospray source of a Fourier transform mass spectrometer, where intact protein ions are fragmented by electron capture dissociation (ECD). The viability of this experimental strategy is demonstrated by applying it to the refolding of horse apo-myoglobin (aMb), a reaction known to involve a transient intermediate. Cooling of the mixing device to 0 °C reduces the reaction rate such that the folding process occurs within the experimentally accessible time window. Top-down ECD provides an average spatial resolution of ca. 2 residues, surpassing the resolution typically achieved in traditional proteolytic digestion/HDX studies. Amide back exchange is virtually eliminated by the short (∼1 s) duration of the acid quenching step. The aMb folding intermediate exhibits HDX protection in helices G and H, whereas the remainder of the protein is largely unfolded. Marginal protection is seen for helix A. Overall, the top-down ECD approach used here offers insights into the sequence of events leading from the unfolded state to the native conformation, with envisioned future applications in the areas of protein misfolding and aggregation. The time-resolved experiments reported herein represent an extension of our previous work, where HDX/MS with top-down ECD was employed for monitoring static protein structures under equilibrium conditions (Pan et al. J. Am. Chem. Soc. 2009, 131, 12801)

Carotenoid and pheophytin on semiconductor surface: Self-assembly and photoinduced electron transfer

Self-assembling of a carotenoid and pheophytin a into a supramolecular system was observed on the surface of nanocrystalline TiO2, and the photoinduced electron-transfer reactions within the system were studied by means of femtosecond transient absorption and laser flash photolysis techniques. Excitation of the pheophytin moiety results in ultrafast electron transfer from carotenoid to the excited pheophytin, creating a long-lived charge-separated state. Two decay pathways of the formed pheophytin a anion radical are proposed. The first is a direct back electron recombination forming a carotenoid triplet state on the nanosecond time scale, while the other is suggested to occur via electron injection to the TiO2 nanoparticle. These results demonstrate that a self-assembled carotenoid-pheophytin system leads to an efficient reductive quenching of the pheophytin moiety, suggesting that a similar mechanism can operate also in natural photosynthetic systems. Moreover, the formation of a long-lived charge-separated state indicates that such self-assembling strategy may be also considered for novel dye-sensitized solar cell constructions and other artificial systems aiming to mimic the electron-transfer chain in natural photosynthesis

Solution-Phase Chelators for Suppressing Nonspecific Protein−Metal Interactions in Electrospray Mass Spectrometry

Protein-metal complexes may be transferred from solution into the gas phase by electrospray ionization (ESI), such that they can be directly analyzed by mass spectrometry (MS). In principle, therefore, ESI-MS represents a simple and elegant approach for gaining insights into the binding stoichiometry and affinity of these assemblies. Unfortunately, the formation of nonspecific metal adducts during ESI can be a severe problem, often leading to binding levels that are dramatically higher than those in bulk solution. Focusing on several calcium binding proteins as test systems, this work explores the suitability of different salts to serve as metal source. Despite their widespread use in previous ESI-MS studies, calcium chloride and acetate induce extensive nonspecific adduction. In contrast, much lower levels of artifactual metal binding are observed in the presence of calcium tartrate. In the case of high and intermediate affinity proteins, the resulting ESI-MS data are in excellent agreement with the calcium binding behavior in bulk solution. The situation is more challenging when studying proteins with very low affinities, but in the presence of tartrate qualitative information on protein-metal interactions can still be obtained. The beneficial effects of tartrate also extend to zinc binding experiments. This work does not directly explore the mechanism by which tartrate suppresses nonspecific metalation. However, it seems likely that weak chelators such as tartrate sequester metal ions within rapidly shrinking droplets during the final stages of ESI, thereby reducing nonspecific metal adduction to protein carboxylates. The use of tartrate and possibly other weak chelators will greatly enhance the reliability of future ESI-MS studies on the interactions of proteins with divalent metal ions

Excited-state processes in the carotenoid zeaxanthin after excess energy excitation

Aiming for better understanding of the large complexity of excited-state processes in carotenoids, we have studied the excitation wavelength dependence of the relaxation dynamics in the carotenoid zeaxanthin. Excitation into the lowest vibrational band of the S-2 state at 485 nm, into the 0-3 vibrational band of the S2 state at 400 nm, and into the B-2(u)+ state at 266 nm resulted in different relaxation patterns. While excitation at 485 nm produces the known four-state scheme (S-2 -> hot S-1 -> S-1 -> S-0), excess energy excitation led to additional dynamics occurring with a time constant of 2.8 ps (400 nm excitation) and 4.9 ps (266 nm excitation), respectively. This process is ascribed to a conformational relaxation of conformers generated by the excess energy excitation. The zeaxanthin S* state was observed regardless of the excitation wavelength, but its population increased after 400 and 266 nm excitation, suggesting that conformers generated by the excess energy excitation are important for directing the population toward the S* state. The S-2-S-1 internal conversion time was shortened from 135 to 70 fs when going from 485 to 400 nm excitation, as a result of competition between the S-2-S-1 internal conversion from the vibrationally hot S2 state and S2 vibrational relaxation. The S, lifetime of zeaxanthin was within experimental error the same for all excitation wavelengths, yielding similar to 9 ps. No long-lived species have been observed after excitation by femtosecond pulses regardless of the excitation wavelength, but excitation by nanosecond pulses at 266 nm generated both zeaxanthin triplet state and cation radical

Subzero Temperature Chromatography and Top-Down Mass Spectrometry for Protein Higher-Order Structure Characterization: Method Validation and Application to Therapeutic Antibodies

Characterization of the higher-order structure and structural dynamics of proteins is crucial for in-depth understanding of their functions. Amide hydrogen/deuterium exchange (HDX), monitored by mass spectrometry (MS), is now a popular technique for measuring protein higher-order structural changes. Although the proteolysis-based HDX-MS approach is most commonly used, the “top-down” approach, which fragments intact proteins directly using electron-based dissociation, is becoming an important alternative and has several advantages. However, the commonly used top-down strategies are direct-infusion based and thus can only be used with volatile buffers. This has meant that the “top-down” approach could not be used for studying proteins under physiological conditionsthe very conditions which are often very important for preserving a protein’s native structure and function. More complex proteins such as those with disulfide bonds present another challenge. Therefore, there is significant interest in developing novel top-down HDX methods that are applicable to all types of protein samples. In this paper, we show how top-down electron capture dissociation and subzero temperature HPLC can be combined and used for this purpose. This method keeps the back-exchange level as low as 2% and has no limitations in terms of protein type and sample solution conditions. Close to single-residue level protein structural information can be generated. The new method is validated through comparison with NMR data using calmodulin as a model protein. Its capability of determining structural changes in therapeutic antibodies (Herceptin) is also demonstrated

Stepwise charge separation from a ruthenium-tyrosine complex to a nanocrystalline TiO2 film

A supramolecular complex composed of Ru(II) tris-bipyridine, tyrosine, and dipicolylamine was synthesized and characterized. This complex was attached to TiO2 nanocrystalline films via ester groups at the Ru(II) chromophore, and photoinduced multistep electron transfer was investigated by laser flash photolysis and electron paramagnetic resonance techniques. Following ultrafast electron injection from the metal-ligand charge transfer excited states of Ru(II) to the conduction band of TiO2, fast intramolecular electron transfer from the tyrosine moiety to the photogenerated Ru(III) was observed, characterized by a rate constant of similar to2 x 10(6) s(-1). By comparison of recovery kinetics at the isosbestic point with that of the reference compound lacking the tyrosine, it was found that the intramolecular electron-transfer efficiency is 90%. A hydrogen-bond-promoted electron-transfer mechanism is proposed

Anthraquinone dyes as photosensitizers for dye-sensitized solar cells

Three anthraquinone dyes with carboxylic acid as anchoring group are designed and synthesized as sensitizers for dye-sensitized solar cells (DSSCs). Preliminary photophys. and photoelectrochem. measurements show that these anthraquinone dyes have very low performance on DSSC applications, although they have broad and intense absorption spectra in the visible region (up to 800 nm). Transient absorption kinetics, fluorescence lifetime measurements and d. functional theory (DFT) calcns. are conducted to investigate the cause of such low DSSC performance for these dyes. The strong electron-withdrawing character of the two carbonyl groups on anthraquinone framework may lie behind the low performance by suppressing the efficient electron injection from the dye to the conduction band of TiO2