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.

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 OEC’s S2 state. From 17O-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 17O signals incorporated in distinctly different time regimes. First, a signal appearing after two hours of 17O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the 17O 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 17O μ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-μ-oxo position, from which 17O 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 S2 state of the OEC was obtained following multi-hour 17O exchange, which showed a 17O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as μ-oxo-17O by resemblance to the μ-oxo signals from Mn catalase and the di-μ-oxo di-Mn(III,IV) bipyridine model.

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, 1s 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.

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.

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. (14)N- and (15)N-nitrite substrates gave rise to characteristic (14)NO and (15)NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with (63)Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated approximately 50% spin on the N of NO and approximately 17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cdelta1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cdelta1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 A from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).

Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO 2 − + 2H + + e − → NO + H 2 O). 1-3 Recently a side-on bound copper nitrosyl complex of NiR has been crystallized by reacting the reduced enzyme with excess NO. 4 Based on the EPR spectrum of the species generated by the reaction of reduced NiR with saturated NO in solution, it has been assigned as an η 2 -NO − Cu 2+ species. However, spectroscopic data on several Cu-nitrosyl model complexes 5 and an NO adduct formed by reacting reduced NiR with nitrite 6 and DFT calculations on an η 2 -NOCu species 7 describe these complexes as Cu + NO • . In this study, we define the species generated in solution by reacting reduced NiR with NO, using EPR, MCD and ENDOR spectroscopy and correlate these with that observed by crystallography.Reduced NiR reacted with saturated NO solution generates the EPR spectrum in Figure 1A red, which is equivalent to the spectrum reported by Tocheva et al. 4 This species has an EPR signal characteristic of an oxidized T2 Cu (g || = 2.30 and A || = 117 G). The MCD spectrum (Fig. 1B, red) has low energy d-d bands, also indicative of a T2 cupric complex. Both EPR and MCD spectra show a negligible T1 contribution indicating that this site is reduced (Fig. S1).Nitrite binds to the T2 site of the resting WT enzyme, 8,9 but the EPR and, in particular, the MCD features of the nitrite bound T2 site are obscured by the dominant T1 Cu signals. However under these conditions (nitrite bound), the T1 Cu could be selectively reduced with acsorbate. The EPR spectrum of the nitrite bound T2 species (Fig. 1A, blue) is identical to that produced above by reacting reduced NiR with excess NO. The MCD spectrum (Fig. 1B, blue) of the T1 reduced T2-nitrite bound form is also identical to that generated by the reduced enzyme with NO. The T2 EPR spectrum and the MCD d-d bands of the resting WT enzyme are very sensitive to nitrite binding (Fig. S1C, D). Thus the EPR and MCD data strongly indicate that the species generated by the reaction of excess NO with reduced NiR has T1 reduced and T2 oxidized with nitrite bound.Further N 14 and H 1 ENDOR data for this species generated by the reaction of reduced NiR with an NO solution (Fig. 1C, D, red) and those for a form with T1+T2 oxidized and nitrite bound (collected at g=2.266 where there is no contribution from T1, Fig. 1C, D, blue) have the same features. The nitrogen features (Fig. 1C) From the above results the reaction of reduced NiR with excess NO gives T1 reduced T2 Cu 2+ -NO 2 − . However, the reaction of the reduced enzyme with limited NO has an EPR signal ( Fig. 2A, green), characteristic of a Cu + NO • species. 6 This species lacks d-d bands in the MCD spectrum (Fig. S3), further confirming that the Cu is reduced. Therefore, reduced NiR forms a Cu + NO • species at low concentrations of NO.The reaction of reduced NiR with excess NO involves two one-electron oxidati...

The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio or=3:1 complex also required intact Zn fingers. Stopped-flow EPR kinetics with NCP7/RNA mixed at a 4:1 ratio showed the major phase of NCp7 interaction with RNA stem loop 3 occurred within 4 ms, a second phase occurred with a time constant of approximately 30 ms, and a slower immobilization, possibly concomitant with large complex formation, proceeded over seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for viral packaging and genome fabrication.

We have developed a variable velocity, rapid-mix, continuous-flow method for observing and delineating kinetics by dielectric resonator-based electron paramagnetic resonance (EPR). The technology opens a new facet for kinetic study of radicals in liquid at submillisecond time resolution. The EPR system (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instrum. 65:68-74) accommodated a miniature quartz capillary mixer with an approximately 0.5 microliter delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum delivery time of approximately 150 microseconds. The mixing was efficient, and we constructed kinetics in the 0.15-2. 1-ms time range by plotting the continuous wave EPR signal taken during flow versus the reciprocal of flow velocity. We followed the refolding kinetics of iso-1-cytochrome c spin-labeled at Cysteine 102. At 20 degrees C, upon dilution of guanidinium hydrochloride denaturant, a fast phase of refolding was resolved with an exponential time constant of 0.12 ms, which was consistent with the "burst" phase observed by optically detected flow techniques. At 7 degrees C the kinetic refolding time of this phase increased to 0.5 ms.