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The primary electron donor P700 of the photosystem I (PSI) is a heterodimer consisting of two chlorophyll molecules. A series of electron transfer events immediately following the initial light excitation leads to a stabilization of the positive charge by its cation radical form, . The electronic structure of and, in particular, its asymmetry with respect to the two chlorophyll monomers is of fundamental interest and is not fully understood up to this date. Here, we apply multi-frequency X-(9 GHz) and Q-band (35 GHz) hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate the electron spin density distribution in the cation radical of PSI from a thermophilic cyanobacterium Thermosynechococcus elongatus. Six 14 N and two 1 H distinct nuclei have been resolved in the HYSCORE spectra and parameters of the corresponding nuclear hyperfine and quadrupolar hyperfine interactions were obtained by combining the analysis of HYSCORE spectral features with direct numerical simulations. Based on a close similarity of the nuclear quadrupole tensor parameters, all the resolved 14 N nuclei were assigned to six out of total eight available pyrrole ring nitrogen atoms (i.e., four in each of the chlorophylls), providing the direct evidence of spin density delocalization over the both monomers in the heterodimer. Using the obtained experimental values of the 14 N electronnuclear hyperfine interaction parameters, the upper limit of the electron spin density asymmetry parameter is estimated as while a tentative assignment of 14 N observed in the HYSCORE spectra yields .

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We introduced GXXXG motifs in a pair of de-novo designed (AALALAA) 3 helices incorporated into POPC liposomes with their topology controlled in a parallel fashion, and their self-association was monitored by single-pair FRET in real time. The introduction of a GXXXG sequence in the middle of the sequence (AALALAA AGLALGA AALALAA) facilitated the dimerization of helices, which was abolished by cholesterol. In contrast, the incorporation of a longer GXXXGXXXG segment (AALALAA AGLALGA AAGALAA) promoted the self-association of helices in POPC/cholesterol bilayers, but not without cholesterol. The inclusion of a longer motif (AALALGA AGLALGA AGLALAA) nullified the dimerization. Furthermore, the dimerization of GXXXG-introduced helices was found highly sensitive to amino acid sequences flanking the GXXXG motif and its position along the entire sequence. Underlining mechanisms will be discussed.

Liposomes have important applications for drug delivery. Whether asymmetric large unilamellar vesicles (asymmetric LUVs) with different charges on different leaflets can be efficiently prepared and have practical applications have advantages for efficient drug encapsulation and efficiency has not been explored. To examine this we have developed methods to prepare asymmetric LUV with opposite charges on the inner and outer leaflet. We investigated liposomes mixtures containing one of three different kinds of negative charged lipids, POPG, POPS and POPA, one of two different kinds of positive charged lipids, POePC and DOTAP, and a zwitterionic lipid, POPC. Using the cyclodextrin (CD) assisted exchange methods developed by our group, we have prepared asymmetric LUVs with about 25 % of POPS or POPA in the outer or inner leaflet (monolayer) and about 25 % of POePC or DOTAP on the opposite leaflet, with about 75 % of lipids in each leaflets being POPC. Exchange with POPG was somewhat less efficient. To investigate asymmetry of the LUVs a TMA-DPH binding assay was developed. Future studies will investigate drug entrapment ability and the efficiency of drug delivery using these liposomes.