Polycyclic aromatic hydrocarbons (PAHs) are widespread hazardous, hydrophobic pollutants made of fused benzene rings. Besides of being present in large quantities in petroleum and related products they are formed generally during the incomplete combustion of organic matter. During evolution, bacteria have become capable of using such recalcitrant compounds as growth substrates. Hundreds of species of bacteria have been identified that can metabolise a wide range of contaminants, being of potential use in bioremediation strategies. The types of molecules that can be degraded depend on the metabolic pathway in each species. The genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The a and b subunits of a dioxygenase complex (FlnA1A2), showing 63% and 51% sequence identity respectively, with the subunits of an angular dioxygenase from the Gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in E. coli, FlnA1A2 was responsible for the angular oxidation of fluorene, fluorenol, fluorenone, dibenzofuran and dibenzo-p-dioxin. Moreover, FlnA1A2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. Quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1A2. Sphingomonas sp. strain LH128 harbors a naphthalene dioxygenase (PhnA1A2f) responsible for the oxidation of PAHs made of up to four rings, monochlorinated biphenyls and dibenzo-p-dioxin. PhnA1A2f shows exceptionally broad substrate specificity towards various pollutants in contrast to other naphthalene dioxygenases whose activity is limited to two- and three-ring PAHs. Moreover a conserved catabolic gene cluster could be isolated harboring 13 genes involved in PAH degradation. Sequence comparison with the initial dioxygenase of Sphingomonas sp. strain CHY-1 showed that the amino acids lining the catalytic pocket are conserved. The variation in substrate specificity of the two dioxygenases demonstrates that substitutions outside of the catalytic pocket can have marked effects on the substrate range of dioxygenase enzymes.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 6 uppsatser.

Bioremediacija suprantama kaip aplinką valanti technologija, kuri teršalų šalinimui naudoja biologinį potencialą. Apžvalgoje nagrinėjami įvairių polimerinių ir natūralios kilmės junginių biodegradacijos procesų ypatumai, mikrobiologinės grunto bioremediacijos efektyvumo priklausomybė nuo įvairių abiotinių ir biotinių veiksnių. Pateikta analizė apie žemiausios trofinės grandies-mikroorganizmų asociacijų formavimąsi ir funkcionavimą užterštame grunte, aptariamas bioaugmentacijos, biostimuliacijos, biopreparatų efektyvumas naftos produktais užteršto grunto bioremediacijai. Aptariamas naujas ištirtas termofilinių bakterijų naftaleno skaidymo kelias. Gautų duomenų pagrindu apžvalgoje daromos išvados, kad mikroorganizmai gali būti sėkmingai naudojami valant stipriai užterštą gruntą ex-situ sąlygomis. Bioremediacijos technologijos suteikia galimybes pagausinti vietines mikroorganizmų populiacijas, sustiprinti mikroorganizmų veiklą, naudojant papildomas maisto medžiagas, stimuliuoti aktyvių metabolitų sintezę. / Bioremediation is the application of biological processes for the clean up of pollutants present in the environment. The scope of the review encompasses the following subjects: biodegradation of various polymeric and natural origin compounds; the applications of microorganisms to laboratory scale and field scale soil bioremediation, with a focus on petroleum hydrocarbons; bioaugmentation and characterization of microbial communities; the factors affecting soil bioremediation processes including: availability of microbes, accessibility of contaminants, and a heterogeneous environment; the use of microbial metabolites, such as surfactants to improve availability of contaminants. From this review it can be concluded that microorganisms are an effective ex situ technology that can be used for bioremediation of problematic soils. Bioremediation technology allows for the convenient manipulation and control of several environmental parameters that could lead to enhanced and faster treatment of polluted soils: nutrient source (biostimulation), inocula (bioaugmentation), increased availability of pollutants by inducing biosurfactant production inside the microorganisms, etc. Bioremediation also encompasses ex situ methods like enzyme catalyzed pollutant breakdown. The data on the degradation of naphthalene by thermophilic bacteria via a novel pathway, through protocatechuate are presented.

Several nucleoside natural product antibiotics from Streptomyces sp. and actinomycetes have recently been shown to target bacterial peptidoglycan cell wall biosynthesis by inhibiting the bacterial translocase I (MraY). The biosynthetic gene clusters for A-90289, liposidomycins and caprazamycins revealed a protein with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD). This enzyme (LipL) is a mononuclear, non-heme, Fe(II) dependent α-keto glutarate (α-KG) :uridine monophosphate (UMP) dioxygenase responsible for the net dephosphorylation and two electron oxidation of UMP to uridine-5’-aldehyde. The postulated reaction coordinates involving the activation of the C-5’ center in UMP and the corresponding formation of uridine-5’-aldehyde are modeled on extensive spectroscopic and structural characterizations of TauD. In this dissertation, the postulated radical mechanism for LipL involving the formation of an unstable hydroxylated intermediate is investigated via the characterization of a key product obtained from the reaction of LipL (and its homolog Cpr19) with a synthetically modified surrogate substrate where the bridging phosphoester oxygen in UMP is replaced with a 5’ C-P bond. We further validate our hypothesis by analyzing the reactions of both LipL and Cpr19 with specifically 2H1 – labeled UMP substrate and confirming the expected products via mass spectrometry. In addition, we explore substrate promiscuity of the enzymes and utilize a set of site specific mutants of Cpr19 as means of gaining better insight into the active site residues. Predictive models for Cpr19 and LipL structures are developed by the combination of experimental results and chemical logic.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes / AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.

Aromatic pollutants pose a serious environmental problem. Petroleum and its derivates belong to the most abundant contaminants in the Czech Republic and their sanation is a priority objective for improving the life quality of the population. Bioremediation is a technology taking advantage of the natural capacity of soil and water microbial community to degrade environmental pollutants. Deeper understanding and detailed knowledge on specialized bacterial species, pathways and genes is required for selection, optimization and application of targeted bioremediation approach and for monitoring of its results. Recent analysis of a metagenomic library constructed from highly contaminated soil of the former military air-base Hradčany has identified a novel group of catabolic genes encoding extradiol dioxygenase similar to DbtC of Burkholderia sp. DBT1. The DbtC-like enzymes are among the three priority groups of extradiol dioxygenases with biodegradation relevance for the locality. The present study of soil bacterial isolates and metagenomic fosmid clones harboring the genes of interest gained evidence about the dbtC-like genes as a part of highly mobile gene cassette. Transposon insertion mutagenesis identified the genes joined with the expression of the extradiol dioxygenase activity. The dbtC-like genes were...

The former military air-base Hradcany is among the most contaminated with organic pollutants localities in Czech Republic. Main cleanup strategy in the area is the bioremediation taking advantage on the natural potential of the autochthonous soil microorganisms to evolve catabolic pathways for in situ degradation of the pollutant. The diversity and abundance of the pathways, as well as the specificity and activity of the encoded enzymes are priority biotic factors determining the bioremediation efficiency. Main task of this work was to analyze the bacterial diversity in jet fuel contaminated soils based on key catabolic genes encoding the Rieske non-haem iron dioxygenases of the toluene/ biphenyl oxygenase branch. High molecular soil DNA was extracted and the sequences encoding catabolic genes were selectively enriched by hybridization to biotinylated oligonucleotides on magnetic microbeads with covalently bound streptavidin. Fragments of the genes for the -subunits of Rieske non-haem iron oxygenases were amplified and analyzed by restriction analysis, cloning and sequencing. Their evolutionary histories were inferred using the Neighbour-Joining and the maximum likelihood methods. The catabolic genes diversity in the actively bioremediated and highly polluted soil HRB was compared with the diversity in the...

The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis. / Ph. D.

Neste trabalho, usamos a técnica de Ressonância Paramagnética Eletrônica em seu modo convencional para o estudo da enzima clorocatecol 1,2-dioxigenase e em seu modo pulsado para o estudo da estrutura dinâmica de biomembranas. Clorocatecol 1,2-dioxigenase é uma enzima que catalisa a clivagem de estruturas aromáticas, como a do clorocatecol, via a a ativação e incorporação de uma molécula de oxigênio e que possui em seu sítio ativo um íon Fe(III). Nossos resultados de CD e atividade enzimática mostram o intervalo de temperaturas entre 20-25 &ordm;C como aquele no qual a enzima apresenta atividade máxima. A maior contribuição para sua estrutura secundária vem de folhas &#946; anti-paralela. A quantificação do número de spin feita por EPR indicou a presença de um íon Fe(III) por molécula de CCD, estando esse íon em estado de spin alto e simetria rômbica, como se depreende da linha estreita em g=4,3. Os parâmetros de desdobramentos de campo zero foram determinados pela medida em função da temperatura da linha em g=4,3, fornecendo &#955;=E/D=1/3 e D=(1,3&#177;0,2) cm -1. O gráfico de Scatchard construído a partir dos espectros de EPR quando da titulação da enzima com o substrato catecol indicou a presença de um único sítio ligante de catecol, em acordo com a quantificação anterior do metal, e cuja constante de afinidade enzima-substrato é k=(2,7&#177;0,1)x10-6 M. No que tange a EPR pulsado, foram utilizadas as chamadas técnicas modernas de EPR, com ênfase em 2D-ELDOR, para a investigação de diversos aspectos em membranas de interesse biológico. Primeiramente, investigamos a diferenciação entre a fase de líquido ordenado (Lo) e a de líquido cristalino (Lc) em membranas modelo de lipídio puro e lipídio/colesterol (1/1) contendo diferentes marcadores de spin (16-PC, CSL e DPPTC). Aí mostramos que é possível distinguir-se diferentes fases lipídicas apenas por simples inspeção visual dos espectros de 2D-ELDOR de um determinado marcador de spin incorporado à membrana. Além disso, realizamos simulações através do pacote de programas NLSPMC e determinamos as diferenças quantitativas entre as fases lipídicas: o estado de líquido ordenado apresenta maior fluidez e ordenamento na região das cadeias acílicas, ao passo que, na região da cabeça polar, mostra menor ordenamento. Em seguida, estudamos o efeito da presença de colesterol em alta concentração sobre o comportamento da membrana como função da temperatura. Nesse caso, o colesterol atua de maneira a manter a membrana em uma fase altamente ordenada e fluída dentro do intervalo de temperaturas medido, abolindo a transição gel-Lc do lipídio DPPC. Uma tentativa de aplicação de nossa metodologia ao estudo de membranas biológicas foi feita ao estudarmos membranas bleb que são ricas em colesterol. Estas mostram comportamento semelhante àquele observado para as membranas modelo com alta concentração de colesterol, um indicativo da existência de domínios Lo naquelas membranas biológicas. Por fim, estudamos o efeito da presença do peptídeo Gramicidina A\' (GA) sobre a estrutura da membrana de DPPC. Os resultados de 2DELDOR mostram claramente a presença de duas populações de lipídios (\"boundary\" e \"bulk\"). As simulações desses espectros foram as primeiras feitas com espectros contendo duas componentes e com os dados nas formas Sc- e Secsy. Essas mostram que os lipídios \"bulk\" são pouco afetados pela presença de moléculas de GA, ao passo que os lipídios \"boundary\" têm suas cadeias acílicas dobradas na porção terminal em torno das moléculas de GA, mecanismo que suporta a formação de domínios em fase HII na membrana. / In this work, EPR-CW and 2D-FT-EPR are used to study the enzyme chlorocatechol 1,2-dioxygenase and the dynamic structure of biological relevant membranes, respectively. Chlorocatechol 1,2-dioxygenase (CCD) is a non-heme Fe(lIl) enzyme that catalyses the ring cleavage of aromatic compounds like chlorocatechol. The structure stability as a function of the temperature was determined via circular dichroism and catalytic activity assays. The enzyme has its maximum activity at 20-25 &ordm;C. The main contribution to its secondary structure comes from antiparallel &#914; sheets. The iron content was determined by EPR measurements, indicating the presence of one Fe(llI) per molecule. The Fe(III) ion shows a very narrow line at g=4.3 indicating a high spin state in a rhombic symmetry with &#955;=E/D=1/3 and D=(1,3&#177;0,2) cm -1. The Scatchard plot based on EPR spectra suggests the existence of one site for substrate binding with a binding constant k=(2,7&#177;0,1)x10-6 M. As for 2D-FT-EPR, the so-called modem EPR techniques, like 2D-ELDOR, were used to investigate several aspects of biologically relevant membranes. Firstly, we determined the differences between the liquid-ordered (Lo) and the liquid-crystalline (Lc) phases in model membranes of pure lipid and of lipid/cholesterol (1/1) mixtures containing different spin labels (16-PC, CSL, and DPPTC). In this case, we show how 2D-ELDOR makes possible the differentiation between Lo and Lc phases just by a pattern recognition scheme. We also performed simulations of those spectra and the results are: the Lo phase shows higher fluidity and ordering in the acyl chain region, whereas it shows lower ordering in the headgroup polar region. After that the membrane behaviour as s function of temperature was studied. We showed that cholesterol maintains the membrane in a highly ordered and fluid structure over the entire range of temperatures, abolishing the gel-Lc phase transition of the lipid DPPC. The biological membrane bleb was the first attempt to apply our methodology to real membranes. The 2D-ELDOR results for bleb membranes indicate the existence of Lo domains in the structure of those membranes. Finally, we studied the effects of the peptide Gramicidin A\' (GA) on the lipid organization of DPPC membranes. The 2D-ELDOR results show very clear two-component spectra (assigned to bulk and ,boundary lipids), which were simulated by the NLSPMC programs. This is the first time that 2D-FT-EPR multi-component spectra are simulated using both the Sc- and Secsy formal. The simulations indicate that the GA molecules do not significantly affect the bulk lipid, whereas the boundary lipids present the end portion of their acyl chain bent towards the GA molecule. This mechanism is probably responsible for the local formation of the HII phase in the membranes.