The modes of drug–particle interactions considerably influence drug delivery by nanoparticulate carrier systems and drug penetration into the skin. The exact mechanism of the drug loading and its ...release are still ambiguous. Therefore, the loading process, the interaction of the agent and the lipid matrix of solid lipid nanoparticles (SLNs) as well as the uptake of the loaded agent by skin lipids were analysed by electron spin resonance (ESR) and parelectric spectroscopy (PS) using spin probes (TEMPO, TEMPOL, and CAT-1) as model drugs differing in their lipophilicity. The spin probes were closely attached to the particles lipid surface (TEMPO) or located in the layers of the surfactant (CAT-1), respectively. Furthermore, two distinct sub-compartments on the SLN were found. To simulate the processes at the phase boundary SLN dispersion/skin, skin lipid mixtures were prepared and the transfer process of the spin labels was followed by ESR tomography. Transfer rates were related to the lipophilicity of the spin probe, the lipid mixture and the applied pharmaceutical formulation, SLN dispersion and aqueous solution, respectively. In particular, SLN accelerated in particular the distribution of the lipophilic agents.
The fluorescence emission of individual photosystem I complexes from Synechocystis PCC 6803 in protonated and deuterated buffer shows zero-phonon lines as well as broad intensity distributions. The ...number and the line width of the zero phonon lines depend strongly on the solvent (H2O/D2O). The spectral diffusion rate of the whole fluorescence emission from photosystem I is significantly reduced upon deuteration of the solvent. This leads to a substantial increase of well-resolved zero-phonon lines. Since the chlorophyll a chromophores lack exchangeable protons, these observed changes in the spectral diffusion have to be assigned to exchangeable protons at the amino acids and structural water molecules in the chromophore binding pocket.
A quick switch: A single amino acid substitution at a conserved residue (D396N) of Arabidopsis cryptochrome‐1 (Atcry1) confers single‐stranded DNA repair activity in vitro, conferring photolyase ...activity onto the cryptochrome (see graph). The mutant protein undergoes photoreduction of flavin to the fully reduced anionic form, similar to photolyases and unlike wild‐type cryptochromes.
Exploring protein−cofactor interactions on a molecular level is one of the major challenges in modern biophysics. Based on structural data alone it is rarely possible to identify how subtle ...interactions between a protein and its cofactor modulate the protein’s reactivity. In the case of enzymatic processes in which paramagnetic molecules play a certain role, EPR and related methods such as ENDOR are suitable techniques to unravel such important details. In this contribution, we describe how cryogenic-temperature ENDOR spectroscopy can be applied to various LOV domains, the blue-light sensing domains of phototropin photoreceptors, to gain information on the direct vicinity of the flavin mononucleotide (FMN) cofactor by analyzing the temperature dependence of methyl-group rotation attached to C(8) of the FMN’s isoalloxazine ring. More specifically, mutational studies of three amino acids surrounding the methyl group led to the identification of Asn425 as an important amino acid that critically influences the dark-state recovery of Avena sativa LOV2 domains. Consequently, it is possible to probe protein−cofactor interactions on a sub-angstrom level by following the temperature dependencies of hyperfine couplings.
Pulsed electron nuclear double resonance (ENDOR) spectroscopy at Q- and W-band frequencies was applied to single crystals of photosystem II from Th. elongatus. W-Band (1)H-ENDOR on the dark-stable ...radical state Y of the redox-active tyrosine residue Y(D) yields a complete mapping of the electronic structure of this amino acid radical in terms of an assignment of all hyperfine coupling tensors of the protons covalently bound to the side chain. This study can serve as a model case for the potential of high-field/high-frequency ENDOR on protein single crystals for obtaining highly resolved electronic structure information. Q-band (55)Mn-ENDOR was applied to the S(2) oxidation state of the water-splitting complex in photosystem II single crystals. Irrespective of the difficulties arising from the extremely broad electron paramagnetic resonance (EPR) spectroscopy ( approximately 200 mT) and ENDOR ( approximately 100 MHz) spectra a tentative assignment of the Mn ion in the formal oxidation state III to a Mn position in the structural model of PSII is possible on the basis of the ENDOR data.
Hsp70 chaperones assist in a large variety of protein‐folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of ...Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C‐terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure‐based EPR spin‐labelling studies as well as cross‐linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.
The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the ...flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV−vis spectroscopy at low temperatures (T ≤ 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV−vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.
The catalytic center of the NiFe hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron-nuclear double resonance ...spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the beta-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH- bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the "ready" state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the "unready" state of the enzyme.
Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with ...Fe2+ and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.
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