Polar Lipid Fraction E from Sulfolobus acidocaldarius and Dipalmitoylphosphatidylcholine Can Form Stable yet Thermo-Sensitive Tetraether/Diester Hybrid Archaeosomes with Controlled Release Capability.

Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from -48 mV at 37 °C to -16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid 'flip-flop'. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell's cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42-44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions.

GDGT cyclization proteins identify the dominant archaeal sources of tetraether lipids in the ocean.

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are distinctive archaeal membrane-spanning lipids with up to eight cyclopentane rings and/or one cyclohexane ring. The number of rings added to the GDGT core structure can vary as a function of environmental conditions, such as changes in growth temperature. This physiological response enables cyclic GDGTs preserved in sediments to be employed as proxies for reconstructing past global and regional temperatures and to provide fundamental insights into ancient climate variability. Yet, confidence in GDGT-based paleotemperature proxies is hindered by uncertainty concerning the archaeal communities contributing to GDGT pools in modern environments and ambiguity in the environmental and physiological factors that affect GDGT cyclization in extant archaea. To properly constrain these uncertainties, a comprehensive understanding of GDGT biosynthesis is required. Here, we identify 2 GDGT ring synthases, GrsA and GrsB, essential for GDGT ring formation in Sulfolobus acidocaldarius Both proteins are radical S-adenosylmethionine proteins, indicating that GDGT cyclization occurs through a free radical mechanism. In addition, we demonstrate that GrsA introduces rings specifically at the C-7 position of the core GDGT lipid, while GrsB cyclizes at the C-3 position, suggesting that cyclization patterns are differentially controlled by 2 separate enzymes and potentially influenced by distinct environmental factors. Finally, phylogenetic analyses of the Grs proteins reveal that marine Thaumarchaeota, and not Euryarchaeota, are the dominant source of cyclized GDGTs in open ocean settings, addressing a major source of uncertainty in GDGT-based paleotemperature proxy applications.

Nonexponential Kinetics of Loop Formation in Proteins and Peptides: A Signature of Rugged Free Energy Landscapes?

The kinetics of loop formation, i.e., the occurrence of contact between two atoms of a polypeptide, remains the focus of continuing interest. One of the reasons is that contact formation is the elementary event underlying processes such as folding and binding. More importantly, it is experimentally measurable and can be predicted theoretically for ideal polymers. Deviations from single exponential kinetics have sometimes been interpreted as a signature of rugged, protein-like, free energy landscapes. Here we present simulations, with different atomistic models, of short peptides with varied structural propensity, and of a structured protein. Results show exponential contact formation kinetics (or relaxation) at long times, and a power law relaxation at very short times. At intermediate times, a deviation from either power law or simple exponential kinetics is observed that appears to be characteristic of polypeptides with either specific or nonspecific attractive interactions but disappears if attractive interactions are absent. Our results agree with recent experimental measurements on peptides and proteins and offer a comprehensive interpretation for them.

Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius.

Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI).

Homepeptide repeats: implications for protein structure, function and evolution.

Analysis of protein sequences from Mycobacterium tuberculosis H37Rv (Mtb H37Rv) was performed to identify homopeptide repeat-containing proteins (HRCPs). Functional annotation of the HRCPs showed that they are preferentially involved in cellular metabolism. Furthermore, these homopeptide repeats might play some specific roles in protein-protein interaction. Repeat length differences among Bacteria, Archaea and Eukaryotes were calculated in order to identify the conservation of the repeats in these divergent kingdoms. From the results, it was evident that these repeats have a higher degree of conservation in Bacteria and Archaea than in Eukaryotes. In addition, there seems to be a direct correlation between the repeat length difference and the degree of divergence between the species. Our study supports the hypothesis that the presence of homopeptide repeats influences the rate of evolution of the protein sequences in which they are embedded. Thus, homopeptide repeat may have structural, functional and evolutionary implications on proteins.

Oral peptide delivery by tetraether lipid liposomes.

The aim of this study is to improve of oral peptide delivery by a novel type of liposomes containing tetraether lipids (TELs) derived from archaea bacteria. Liposomes were used for the oral delivery of the somatostatin analogue octreotide. TELs were extracted from Sulfolobus acidocaldarius and subsequently purified to single compounds. Liposomes were prepared by the film method followed by extrusion. Vesicles in size between 130 and 207 nm were obtained as confirmed by photon correlation spectroscopy. The pharmacokinetics of radiolabeled TELs in liposomes was investigated after oral administration to rats. 1.6% of the applied radioactivity in fed and 1.5% in fasted rats was recovered in the blood and inner organs after 2h, while most of the radioactivity remained in the gastro-intestinal tract. After 24h the percentage of radioactivity in inner organs was reduced to 0.6% in fed rats, respectively 1.0% in fasted animals. Several liposomal formulations containing dipalmitoyl phosphatidylcholine (DPPC) and TELs in different ratios were loaded with octreotide and orally administered. Liposomes with 25% TEL could improve the oral bioavailability of octreotide 4.1-fold and one formulation with a cationic TEL derivative 4.6-fold. TEL-liposomes probably act by protecting the peptide in the gastro-intestinal tract.

Investigation of archaeosomes as carriers for oral delivery of peptides.

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution.

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.

Formation of a linear [3Fe-4S] cluster in a seven-iron ferredoxin triggered by polypeptide unfolding.

Cubic iron-sulfur ([Fe-S]) clusters are common inorganic cofactors in proteins. The presence of a linear [3Fe-4S] cluster in a protein was first observed in beef-heart aconitase at high pH, where the protein structure was perturbed. Not long ago, the same linear cluster was discovered upon unfolding of a thermophilic di-cluster seven-iron ferredoxin, suggesting a more general relevance for this type of linear clusters in Nature. Since structure-induced cluster rearrangements may be important regulatory, on-going processes in living systems, we decided to further characterize the formation of the linear iron-sulfur cluster observed upon ferredoxin unfolding. Here we present a kinetic investigation of parameters that affect the linear-cluster formation and disassembly in the Sulfolobus acidocaldarius seven-iron ferredoxin. We find the linear cluster to be an intermediate on the protein-mediated cluster-degradation pathway under a wide range of pH and denaturant conditions. The linear species forms in parallel with secondary-structure disappearance. In contrast, the disassembly rate constant for the linear cluster is independent of denaturant concentration but depends strongly on solution pH. At high pH, the disassembly rate is slower and the linear iron-sulfur species has a longer lifetime, than at low pH.

Characterisation of oxidised 7Fe dicluster ferredoxins with NMR spectroscopy.

Dicluster ferredoxins (Fds) from Sulfolobus acidocaldarius and Desulfovibrio africanus (FdIII) have been studied using 1H NMR. Both wild-type proteins contain a [3Fe-4S]+/0 and a [4Fe-4S]2+/+ cluster as isolated. The [4Fe-4S]2+/+ cluster (cluster II) is bound by cysteine residues arranged in a classic ferredoxin motif: CysI-(Xaa)2-CysII-(Xaa)2-CysIII-(Xaa)n-CysIV-Pro , whilst the binding motif of the [3Fe-4S]+/0 cluster (cluster I) has a non-ligating aspartic acid (Asp14) at position II, i.e. CysI-(Xaa)2-Asp-(Xaa)2-CysIII. D. africanus FdIII undergoes facile cluster transformation from the 7Fe form to the 8Fe form, but S. acidocaldarius Fd does not. Many factors determine the propensity of a cluster to undergo interconversion, including the presence, and correct orientation, of a suitable ligand. We have investigated this using 1H NMR by introducing a potential fourth ligand into the binding motif of cluster I of D. africanus FdIII. Asp14 has been mutated to cysteine (D14C), glutamic acid (D14E) and histidine (D14H). Cluster incorporation was performed in vitro. The cluster types present were identified from the chemical shift patterns and temperature-dependent behaviour of the hyperfine-shifted resonances. Factors influencing cluster ligation and cluster interconversion, in vitro, are discussed. Furthermore, the data have established that the residue at position II in the cluster binding motif of cluster I is influential in determining the chemical shift pattern observed for a [3Fe-4S]+ cluster when a short/symmetric binding motif is present. Based on this, a series of rules for characterising the 1H NMR chemical shifts of mono- and di-cluster [3Fe-4S]+ cluster-containing ferredoxins is given.

Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2FE-2S] cluster in Escherichia coli.

The Rieske protein II (Schmidt et al., 1996, FEBS Lett. 388, 43-46) from the thermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was expressed in E. coli cells. The full length protein was strictly bound to the E. coli membranes and could only be removed by detergent treatment indicating the presence of a membrane anchor. The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids. The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein. The midpoint potential was +375 mV determined by CD redox potentiometry. The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.

Identification of the iron-sulfur clusters in a ferredoxin from the archaeon Sulfolobus acidocaldarius. Evidence for a reduced [3Fe-4S] cluster with pH-dependent electronic properties.

A ferredoxin isolated from the archaeon Sulfolobus acidocaldarius strain DSM 639 has been shown to contain one [3Fe-4S]1 + 10 cluster with a reduction potential of -275 mV and one [4Fe-4S]2+/1+ cluster with a reduction potential of -529 mV at pH 6.4, in the temperature range 0-50 degrees C. The monomer molecular mass was confirmed to be 10907.5 +/- 1.0 Da by electrospray mass spectrometry, as calculated from the published amino acid sequence [Minami, Y. Wakabayashi. S., Wada, K., Matsubara, H., Kerscher, L. & Oesterhelt, D. (1985) J. Biochem. (Tokyo) 97, 745-751], while the holoprotein molecular mass was found to be 11,550 +/- 1.0 Da. The reduced [3Fe-4S]0 cluster was also shown by direct electrochemistry and magnetic circular dichroic spectroscopy to undergo a one-proton uptake reaction as first observed for Azotobacter chroococcum ferredoxin I [George, S. J., Richards, A. J. M., Thomson, A. J. & Yates, M. G. (1984) Biochem. J. 224, 247-251]. The pKa of the protonation step has been determined by a novel thin film electrochemical method to be 5.8. This is significantly different from the pKa of 7.7 determined for A. vinelandii ferredoxin I [Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, J. M., Stephens, P. J., LaMar, G. N., Gorst, C. M. & Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939] and indicates that the polypeptide chain around the [3Fe-4S] cluster controls this reaction. Although this appears to be only the second reported case of protonation at or near the reduced [3Fe-4S]0 cluster, its observation in S. acidocaldarius ferredoxin raises the question of the generality of this chemistry for 3Fe clusters. The similarity of the pKa to the estimated intracellular pH of S. acidocaldarius strongly suggests a physiological role for this process.

Purification and characterization of the Rieske iron-sulfur protein from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

The previously detected Rieske iron-sulfur protein from the membranes of the thermoacidophile Sulfolobus acidocaldarius [Anemüller, S., et al. (1993) FEBS Lett. 318, 61-64] was purified to electrophoretic homogeneity and the N-terminal amino acids determined. The apparent molecular weight was estimated to be 32 kDa. The reduced protein displays a rhombic EPR spectrum with gxyz = 1.768, 1.895, 2.035. The average g-value of 1.902 is typical for nitrogen ligand-containing clusters. EPR spin quantification and the iron content indicate the presence of one [2Fe-2S] cluster. The purified protein displays ubiquinol cytochrome c reductase activity. The pH optimum of this reaction is temperature dependent and was determined to be pH 7 at 56 degrees C. The results presented in this study clearly prove that the Sulfolobus Rieske protein belongs to the family of the true Rieske iron-sulfur proteins.

Introduction of additional charges as an aid in protein purification: isolation of elongation factor 2 from Sulfolobus acidocaldarius by preparative isoelectric focusing before and after ADP-ribosylation.

Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archae-bacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.