Vladimir M. Grigoryants

Porphyrin–phospholipid liposomes permeabilized by near-infrared light

The delivery of therapeutic compounds to target tissues is a central challenge in treating disease. Externally controlled drug release systems hold potential to selectively enhance localized delivery. Here we describe liposomes doped with porphyrin–phospholipid that are permeabilized directly by near-infrared light. Molecular dynamics simulations identified a novel light-absorbing monomer esterified from clinically approved components predicted and experimentally demonstrated to give rise to a more stable porphyrin bilayer. Light-induced membrane permeabilization is enabled with liposomal inclusion of 10 molar % porphyrin–phospholipid and occurs in the absence of bulk or nanoscale heating. Liposomes reseal following laser exposure and permeability is modulated by varying porphyrin–phospholipid doping, irradiation intensity or irradiation duration. Porphyrin–phospholipid liposomes demonstrate spatial control of release of entrapped gentamicin and temporal control of release of entrapped fluorophores following intratumoral injection. Following systemic administration, laser irradiation enhances deposition of actively loaded doxorubicin in mouse xenografts, enabling an effective single-treatment antitumour therapy.

EPR–ENDOR Characterization of (17O, 1H, 2H) Water in Manganese Catalase and Its Relevance to the Oxygen-Evolving Complex of Photosystem II

The synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (OEC) of photosystem II (PSII). Understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the OEC. Mn catalase in its Mn(III)-Mn(IV) state is a protein model of the OECs S(2) state. From (17)O-labeled water exchanged into the di-μ-oxo di-Mn(III,IV) coordination sphere of Mn catalase, CW Q-band ENDOR spectroscopy revealed two distinctly different (17)O signals incorporated in distinctly different time regimes. First, a signal appearing after 2 h of (17)O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the (17)O μ-oxo hyperfine coupling of the di-μ-oxo di-Mn(III,IV) bipyridine model (Usov, O. M.; Grigoryants, V. M.; Tagore, R.; Brudvig, G. W.; Scholes, C. P. J. Am. Chem. Soc. 2007, 129, 11886-11887), this signal was assigned to μ-oxo oxygen. EPR line broadening was obvious from this (17)O μ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-μ-oxo position, from which (17)O ENDOR showed a smaller 3.8 MHz hyperfine coupling and possible quadrupole splittings, indicating a terminal water of Mn(III). Exchangeable proton/deuteron hyperfine couplings, consistent with terminal water ligation to Mn(III), also appeared. Q-band CW ENDOR from the S(2) state of the OEC was obtained following multihour (17)O exchange, which showed a (17)O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as μ-oxo-(17)O by resemblance to the μ-oxo signals from Mn catalase and the di-μ-oxo di-Mn(III,IV) bipyridine model.

How Charging Corannulene with One and Two Electrons Affects Its Geometry and Aggregation with Sodium and Potassium Cations

Bowl-shaped mono- and dianions are prepared by reduction of corannulene (C(20)H(10), 1) with sodium and potassium metals in the presence of [18]crown-6 ether. Single-crystal X-ray diffraction studies of two sodium salts, [Na(THF)(2)([18]crown-6)](+)[1(-)] (2a) and [Na([18]crown-6)](+)[1(-)] (2b), reveal the presence of naked corannulene monoanions 1(-) in both cases. In contrast, the potassium adduct, [K([18]crown-6)](+)[1(-)] (3), shows an η(2)-binding of the K(+) ion to the convex face of 1(-). For the first time, corannulene dianions have been isolated as salts with sodium, [Na(2)([18]crown-6)](2+)[1(2-)] (4a) and [Na(THF)(2)([18]crown-6)](+)[Na([18]crown-6)](+)[1(2-)] (4b), and potassium counterions, [K([18]crown-6)](2)(+)[1(2-)] (5). Their structural characterization reveals geometry perturbations upon addition of two electrons to a bowl-shaped polyarene. It also demonstrates η(5)- or η(6)-binding of metals to the curved carbon surface of 1(2-), depending on the crystallization conditions. Both mono- and doubly-charged corannulene bowls show the preferential exo binding of Na(+) and K(+) ions in all investigated compounds. Various types of C-H···π interactions are found in the crystals of 2-5. The UV/Vis, ESR, and (1)H NMR spectroscopic studies of 2-5 indicate different coordination environment of corannulene anions in solution, depending on the metal ion.

Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (≤100 μs) samples to those from commonly shock-frozen (slow freeze, 1 s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

Purple Fibrils: A New Type of Protein Chromophore

A purple color is formed during the fibrillation of lysozyme, a well-studied protein lacking a prosthetic group. The application of Raman spectroscopy, electron paramagnetic resonance and UV-vis absorption spectroscopy indicates the formation of a sulfur∴π-bonded radical cation due to the methionine-phenylalanine interaction, which is consistent with a small molecule model reported in the literature. A purple chromophore with characteristic 550 nm absorption is formed due to a specific orientation of the sulfur-centered radical cation and a phenyl ring stabilized by the fibril framework. A specific fibril conformation and the resulting formation of the chromophore are controlled reversibly by varying the pH. This is the first known example of a side chain self-assembled chromophore formed due to protein aggregation.