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The delivery of oligonucleotides across biological barriers is a challenge of unsurpassed significance at the interface of materials science and medicine, with emerging clinical utility in prophylactic and therapeutic vaccinations, immunotherapies, genome editing, and cell rejuvenation. Here, we address the role of readily available branched lipids in the design, synthesis, and evaluation of isoprenoid charge-altering releasable transporters (CARTs), a pH-responsive oligomeric nanoparticle delivery system for RNA. Systematic variation of the lipid block reveals an emergent relationship between the lipid block and the neutralization kinetics of the polycationic block. Unexpectedly, iA21A11, a CART with the smallest lipid side chain, isoamyl-, was identified as the lead isoprenoid CART for the in vitro transfection of immortalized lymphoblastic cell lines. When administered intramuscularly in a murine model, iA21A11-mRNA complexes induce higher protein expression levels than our previous lead CART, ONA. Isoprenoid CARTs represent a new delivery platform for RNA vaccines and other polyanion-based therapeutics.

Addressing the challenge of understanding how cellular interfaces dictate the mechanical resilience and adhesion of archaeal cells, this study demonstrates the role of the surface layer (S-layer) in methanogenic archaea. Using a combination of atomic force microscopy and single-cell force spectroscopy, we quantified the impact of S-layer disruption on cell morphology, mechanical properties, and adhesion capabilities. We demonstrate that the S-layer is crucial for maintaining cell morphology, where its removal induces significant cellular enlargement and deformation. Mechanical stability of the cell surface is substantially compromised upon S-layer disruption, as evidenced by decreased Young's modulus values. Adhesion experiments revealed that the S-layer primarily facilitates hydrophobic interactions, which are significantly reduced after its removal, affecting both cell-cell and cell-bubble interactions. Our findings illuminate the S-layer's fundamental role in methanogen architecture and provide a chemical understanding of archaeal cell surfaces, with implications for enhancing methane production in biotechnological applications.

Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications. Archaea are proposed to degrade short- and long-chain alkanes to CO 2 by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural knowledge of alkyl-CoM reductases and offers fresh ideas about the expected mechanistic and chemical differences among members of this broad family. We conclude with the challenges of the investigation of these particular enzymes, which might one day generate biofuels for our modern society.

A transmembrane proton gradient is generated and maintained by proton pumps in a cell. Metagenomics studies have recently identified a new category of rhodopsin intermediates between type-1 rhodopsins and heliorhodopsins, named schizorhodopsins (SzRs). SzRs are light-driven inward proton pumps. Comprehensive resonance Raman measurements were conducted to characterize the structure of the retinal chromophore in the unphotolyzed state of four SzRs. The spectra of all four SzRs show that the retinal chromophore is in the all- trans and 15- anti configuration and that the Schiff base is protonated. The polyene chain is planar in the center of the retinal chromophore and is twisted in the vicinity of the protonated Schiff base. The protonated Schiff base in the SzRs forms a stronger hydrogen bond than that in outward proton-pumping rhodopsins. We determined that the hydrogen-bonding partner of the protonated Schiff base is not a water molecule but an amino acid residue, presumably an Asp residue in helix G. The present observations provide valuable insights into the inward proton-pumping mechanism of SzRs.

Diphytanoylphosphatidylcholine (DPhPC) is a synthetic phospholipid in which two methyl-branched acyl chains are introduced into the glycerol moiety, mimicking phospholipids of eukaryotic and eubacterial origins. The lipid bilayers of DPhPC reproduce the outstanding physical properties of methyl-branched lipids that occur in archaeal membranes. DPhPC is commonly used as the base lipid in biophysical experiments, particularly for recording ion-channel currents. However, the dynamics of lipid molecules that induces their useful physical properties is still unclear. In this study, we examined the conformation and orientation of the methyl-branched acyl chain of DPhPC in a membrane using 2 H nuclear magnetic resonance (NMR) measurements of the synthetic lipid with a high stereochemical purity and molecular dynamics (MD) simulations. Deuterium-labeled 3',3'-CD 3 ,D-DPhPC ( 2 ) and 7',7'-CD 3 ,D-DPhPC ( 3 ) showed the characteristic quadrupole splitting width in the 2 H NMR spectra, which corresponded to the bent orientation reported for the archaeal lipid PGP-Me [Yamagami, M., et al. (2019) Biochemistry 58 , 3869-3879]. However, MD simulations, which reproduced the 2 H NMR results well, unveiled the unknown features of DPhPC in the membrane; DPhPC has a chain-specific average orientation, where two bent orientations with upward and downward methyl groups occur at positions C3 and C7 of the sn -1 and sn -2 chains of DPhPC, respectively. These MD and NMR results reveal that these two bent orientations define the average orientation of DPhPC for the shallow part of the acyl chains, which is considered to be an important factor in the stability of DPhPC membranes.

Metallopeptidases cleave polypeptides bound in the active-site cleft of catalytic domains through a general base/acid mechanism. This involves a solvent molecule bound to a catalytic zinc and general regulation of the mechanism through zymogen-based latency. Sixty reported structures from 11 metallopeptidase families reveal that prosegments, mostly N-terminal of the catalytic domain, block the cleft regardless of their size. Prosegments may be peptides (5-14 residues), which are only structured within the zymogens, or large moieties (<227 residues) of one or two folded domains. While some prosegments globally shield the catalytic domain through a few contacts, others specifically run across the cleft in the same or opposite direction as a substrate, making numerous interactions. Some prosegments block the zinc by replacing the solvent with particular side chains, while others use terminal α-amino or carboxylate groups. Overall, metallopeptidase zymogens employ disparate mechanisms that diverge even within families, which supports that latency is less conserved than catalysis.

Photoinduced biochemical reactions are ubiquitously governed by derivatives of flavin, which is a key player in a manifold of cellular redox reactions. The photoreactivity of flavins is also one of their greatest disadvantages as the molecules are sensitive to photodegradation. To prevent this unfavorable reaction, UV-light-exposed archaea bacteria, such as Halobacterium salinarum, manage the task of protecting flavin derivatives by dodecin, a protein which stores flavins and efficiently photoprotects them. In this study, we shed light on the photoprotection mechanism, i.e., the excited state quenching mechanism by dodecin using computational methodology. Molecular dynamics (MD) simulations unraveled the hydrogen bond network in the flavin binding pocket as a starting point for proton transfer upon preceding electron transfer. Using high-level ab initio quantum chemical methods, different proton transfer channels have been investigated and an energetically feasible Mg 2+ -promoted channel has been identified fully explaining previous experimental observations. This is the first extensive theoretical study of archaeal dodecin, furthering the understanding of its photocycle and manipulation.

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M(-1) cm(-1)) and 558 nm (2290 M(-1) cm(-1)) in the UV-vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10(-4) cm(-1), and another more rhombic, with a smaller A∥ value of 69 × 10(-4) cm(-1). The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV-vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

The lipid composition of archaea is unique and has been correlated with increased stability under extreme environmental conditions. In this article, we have focused on the evolution of membrane organization and dynamics with natural evolution. Dynamic anisotropy along the membrane normal (i.e., gradients of mobility, polarity, and heterogeneity) is a hallmark of fluid phase diester or diether phospholipid membranes. We monitored gradients of mobility, polarity, and heterogeneity along the membrane normal in membranes made of a representative archaeal lipid using a series of membrane depth-dependent fluorescent probes, and compared them to membranes prepared from a typical diether lipid from higher organisms (eukaryotes). Our results show that the representative dynamic anisotropy gradient along the membrane normal is absent in membranes made from archaeal lipids. We hypothesize that the dynamic gradient observed in membranes of diester and diether phospholipids is a consequence of natural evolution of membrane lipids in response to the requirement of carrying out complex cellular functions by membrane proteins.

In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.

We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes.

Gene therapy for treating inherited diseases like cystic fibrosis might be achieved using multimodular nonviral lipid-based systems. To date, most optimizations have concerned cationic lipids rather than colipids. In this study, an original archaeal tetraether derivative was used as a colipid in combination with one or the other of two monocationic amphiphiles. The liposomes obtained, termed archaeosomes, were characterized regarding lipid self-assembling properties, macroscopic/microscopic structures, DNA condensation/neutralization/relaxation abilities, and colloidal stability in the presence of serum. In addition, gene transfer experiments were conducted in mice with lipid/DNA complexes being administered via systemic or local delivery routes. Altogether, the results showed that the tetraether colipid can provide complexes with different in vivo transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the administration route. This original colipid appears thus as an innovative modular platform endowed with properties possibly beneficial for fine-tuning of in vivo lipofection and other biomedical applications.

Aeropyrum pernix is an aerobic hyperthermophilic archaeon that grows in harsh environmental conditions and as such possesses unique structural and metabolic features. Its membrane interfaces with the extreme environment and is the first line of defense from external factors. Therefore, lipids composing this membrane have special moieties that increase its stability. The membrane of A. pernix is composed predominantly of two polar lipids 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI). Both have methyl branches in their lipid tails and ether linkages and carbohydrates in their headgroup. These moieties significantly affect the structure and dynamics of the bilayer. To provide a molecular level insight into these characteristics, we used here Molecular Dynamics (MD) simulations of lipid bilayers of composition similar to those of the archaeal membranes. First, we show that the electron density profiles along the normal to the bilayers derived from the simulations are in good agreement with the profiles obtained by the small-angle X-ray scattering (SAXS) technique, which provides confidence in the force fields used. Analyses of the simulation data show that the archaeal lipid bilayers are less hydrated than conventional phosphatidylcholine (PC) lipids and that their structure is not affected by the salt present in the surrounding solution. Furthermore, the lateral pressure in their hydrophobic core, due to the presence of the branched tails, is much higher than that at PC-based lipid bilayers. Both the methyl branched tails and the special headgroup moieties contribute to slow drastically the lateral diffusion of the lipids. Furthermore, we found that the lipid head groups associate via hydrogen bonding, which affects their reorientational dynamics. All together, our data provide links between the microscopic properties of these membranes and their overall stability in harsh environments.

Our group recently determined that a mutant archaeal chaperonin (Hsp 60) exhibited substantially enhanced protein folding activity at low temperatures and was able to deconstruct refractory protein aggregates. ATP dependent conversion of fibril structures into amorphous aggregates was observed in insulin amyloid preparations (Kurouski et al. Biochem. Biophys. Res. Commun. 2012). In the current study, mechanistic insights into insulin fibril deconstruction were obtained by examination of early stage complexes between Hsp60 and fibrils in the absence of ATP. Activity of the Hsp60 was significantly curtailed without ATP; however, some fibril deconstruction occurred, which is consistent with some models of the folding cycle that predict initial removal of unproductive protein folds. Chaperonin molecules adsorbed on the fibril surface and formed chaperonin clusters with no ATP present. We propose that there are specific locations on the fibril surface where chaperonin can unravel the fibril to release short fragments. Spontaneous coagulation of these fibril fragments resulted in the formation of amorphous aggregates without the release of insulin into solution. The addition of ATP significantly increased the toxicity of the insulin fibril-chaperonin reaction products toward mammalian cells.

The polar lipid fraction E (PLFE) is a major tetraether lipid component in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Using differential scanning and pressure perturbation calorimetry as well as ultrasound velocity and density measurements, we have determined the compressibilities and volume fluctuations of PLFE liposomes derived from different cell growth temperatures (T(g) = 68, 76, and 81 °C). The compressibility and volume fluctuation values of PLFE liposomes, which are substantially less than those detected from diester lipid membranes (e.g., DPPC), exhibit small but significant differences with T(g). Among the three T(g)s employed, 76 °C leads to the least compressible and most tightly packed PLFE membranes. This temperature is within the range for optimal cell growth (75-80 °C). It is known that a decrease in T(g) decreases the number of cyclopentane rings in archael tetraether lipids. Thus, our data enable us to present the new view that membrane packing in PLFE liposomes varies with the number of cyclopentane rings in a nonlinear manner, reaching maximal tightness when the tetraether lipids are derived from cells grown at optimal T(g)s. In addition, we have studied the effects of pressure on total layer thickness, d, and neutron scattering length density, ρ(n), of a silicon-D(2)O interface that is covered with a PLFE membrane using neutron reflectometry (NR). At 55 °C, d and ρ(n) are found to be rather insensitive to pressure up to 1800 bar, suggesting minor changes of the thickness of the membrane's hydrophobic core and headgroup orientation upon compression only.

The robustness of microorganisms used in industrial fermentations is essential for the efficiency and yield of the production process. A viable tool to increase the robustness is through engineering of the cell membrane and especially by incorporating lipids from species that survive under harsh conditions. Bolalipids are tetraether lipids found in Archaea bacteria, conferring stability to these bacteria by spanning across the cytoplasmic membrane. Here we report on in silico experiments to characterize and design optimal bolalipid membranes in terms of robustness. We use coarse-grained molecular dynamics simulations to study the structure, dynamics, and stability of membranes composed of model bolalipids, consisting of two dipalmitoylphosphatidylcholine (DPPC) lipids covalently linked together at either one or both tail ends. We find that bolalipid membranes differ substantially from a normal lipid membrane, with an increase in thickness and tail order, an increase in the gel-to-liquid crystalline phase transition temperature, and a decrease in diffusivity of the lipids. By changing the flexibility of the linker between the lipid tails, we furthermore show how the membrane properties can be controlled. A stiffer linker increases the ratio between spanning and looping conformations, rendering the membrane more rigid. Our study may help in designing artificial membranes, with tunable properties, able to function under extreme conditions. As an example, we show that incorporation of bolalipids makes the membrane more tolerant toward butanol.

It is known that the thermoacidophilic archaebacterium Sulfolobus acidocaldarius can grow in hot springs at 65-80 degrees C and live in acidic environments (pH 2-3); however, the origin of its unusual thermal stability remains unclear. In this work, using a vesicle as a model, we study the thermal stability and rupture of archaebacterial cell membrane. We perform a simulation investigation of the structure-property relationship of monolayer membrane formed by bolaform lipids and compare it with that of bilayer membrane formed by monopolar lipids. The origin of the unusually thermal stability of archaebacterial cell and the mechanism for its rupture are presented in molecular details.

A synthetic route for the preparation of symmetrical and unsymmetrical archaeal tetraether-like analogues has been described. The syntheses are based upon the elaboration of hemimacrocyclic tetraether lipid cores from versatile building blocks followed by simultaneous or sequential introduction of polar head groups. Functionalizations of the tetraether lipids with neutral lactose or phosphatidylcholine polar heads and cationic glycine betaine moieties were envisaged both to increase membrane stability and to exhibit interactions with charged nucleic acids. Additionally, mannose and lactose triantennary clusters designed as multivalent ligands for selective interaction with lectin-type receptors were also efficiently synthesized for active cell/tissue targeting.

Zinc is one of the metal ions essential for life, as it is required for the proper functioning of a large number of proteins. Despite its importance, the annotation of zinc-binding proteins in gene banks or protein domain databases still has significant room for improvement. In the present work, we compiled a list of known zinc-binding protein domains and of known zinc-binding sequence motifs (zinc-binding patterns), and then used them jointly to analyze the proteome of 57 different organisms to obtain an overview of zinc usage by archaeal, bacterial, and eukaryotic organisms. Zinc-binding proteins are an abundant fraction of these proteomes, ranging between 4% and 10%. The number of zinc-binding proteins correlates linearly with the total number of proteins encoded by the genome of an organism, but the proportionality constant of Eukaryota (8.8%) is significantly higher than that observed in Bacteria and Archaea (from 5% to 6%). Most of this enrichment is due to the larger portfolio of regulatory proteins in Eukaryota.

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.

[reaction: see text] Substituted diphenylamines can be synthesized by Pd(0)-catalyzed N-arylation using o-nitroanilines and nitro-substituted aryl bromides for a substrate. Cyclization of the diphenylamines by various methods, including the intramolecular Pd(0)-catalyzed N-arylation, produces 2-methoxyphenazine which can easily be deprotected to give 2-hydroxyphenazine. This phenazine is required to synthesize methanophenazine, a novel redoxactive cofactor isolated from methanogenic archaea.

The three-dimensional structure of methanogen chromosomal protein 1 (MC1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55, has been solved using (1)H NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an alpha helix and five beta strands, arranged as two antiparallel beta sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two beta sheets and the helix side facing the interior of the protein. The MC1 solution structure shows a globular protein with overall dimensions in the range of 34-40 A, which potentially corresponds to a DNA-binding site of 10-12 base pairs. The presumed DNA-binding site is located on the sequence comprising residues K62-P82, which is formed by a part of strands II2 and II3 belonging to the triple-stranded antiparallel beta sheet and a loop flanked by prolines P68 and P76. The tryptophan W74 that is expected to play a key role in the DNA-binding according to photocross-linking experiments was found completely exposed to the solvent, in a good position to interact with DNA. The overall fold of MC1, characterized by its linking beta-beta-alpha-beta-beta-loop-beta, is different from other known DNA-binding proteins. Its structure suggests a different DNA-binding mode than those of the histone-like proteins HU or HMGB. Thus, MC1 may be classified as a member of a new family.

Genome sequence data of the cold-adapted archaeon, Methanococcoides burtonii, was linked to liquid chromatography-mass spectrometry analysis of the expressed-proteome to define the key biological processes functioning at 4 degrees C. 528 proteins ranging in pI from 3.5 to 13.2, and 3.5-230 kDa, were identified. 133 identities were for hypothetical proteins, and the analysis of these is described separately (Goodchild et al. manuscript in preparation). DNA replication and cell division involves eucaryotic-like histone and MC1-family DNA binding proteins, and 2 bacterial-like FtsZ proteins. Eucaryotic-like, core RNA polymerase machinery, a bacterial-like antiterminator, and numerous bacterial-like regulators enable transcription. Motility involves flagella synthesis regulated by a bacterial-like chemotaxis system. Lsmalpha and Lsmgamma were coexpressed raising the possibility of homo- and hetero-oligomeric complexes functioning in RNA processing. Expression of FKBP-type and cyclophilin-type peptidyl-prolyl cis-trans isomerases highlights the importance of protein folding, and novel characteristics of folding in the cold. Thirteen proteins from a superoperon system encoding proteasome and exosome subunits were expressed, supporting the functional interaction of transcription and translation pathways in archaea. Proteins involved in every step of methylotropic methanogenesis were identified. CO(2) appears to be fixed by a modified Calvin cycle, and by carbon monoxide dehydrogenase. Biosynthesis involves acetyl-CoA conversion to pyruvate by a non-oxidative pentose phosphate pathway, and gluconeogenesis for the conversion of pyruvate to carbohydrates. An incomplete TCA cycle may supply biosynthetic intermediates for amino acid biosynthesis. A novel finding was the expression of Tn11- and Tn12-family transposases, which has implications for genetic diversity and fitness of natural populations. Characteristics of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

Unsymmetrical archaeal tetraether glycolipid analogues 1-2 incorporating a 1,3-disubstituted cyclopentane ring into the bridging chain have been synthesized. The cyclopentane has been introduced with a totally controlled cis configuration, either into the middle of the aliphatic chain or at three methylene groups from the glycerol unit linked to the bulkier disaccharide residue. Freeze-fracture and cryotransmission electron microscopy experiments clearly demonstrated unprecedented glycolipid supramolecular organizations involving two-by-two monolayer associations coupled with interconnection and fusion phenomena. Furthermore, a significant difference in the hydration properties and in the lyotropic liquid crystalline behavior of bipolar lipids 1-2 was found depending on the position of the cyclopentane residue.

Mt0807 is an 85-residue thiol-redox protein from the anaerobic archaebacterium Methanobacterium thermoautotrophicum. Its small size, its participation in certain redox reactions, and the presence of a "classic" glutareodoxin active-site sequence have led to the suggestion that it might be a glutaredoxin. However, studies by previous workers indicated that it exhibited neither glutaredoxin-like nor thioredoxin-like properties. To clarify the true role of this protein and its structure/functional relationship with a paralogous thioredoxin (Mt0895, 28% sequence identity) and a recently characterized orthologous protein (Mj0307, 51% sequence identity), we undertook a series of biochemical and biophysical studies. Comparative enzymatic assays and thiol titration experiments were combined with NMR structural studies and detailed 3D structure comparisons. Structurally, our results show that Mt0807 has a glutaredoxin-like fold (central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other). However, more detailed comparisons with other members of the thioredoxin superfamily indicate that Mt0807 actually has several key structural and active-site characteristics more common to a thioredoxin. Furthermore, biochemical tests show that Mt0807 actually behaves as true thioredoxin. Comparisons between Mt0807 and its paralogue, Mt0895, indicate these two archaebacterial thioredoxins share very similar folds, but exhibit very different activities and likely serve somewhat different roles. On the basis of its greater relative abundance and significantly stronger redox activity, we believe that Mt0807 is the primary thioredoxin for M. thermoautotrophicum, while Mt0895 plays a minor or supportive role. We also suggest that these two molecules (Mt0807 and Mt0895) may represent a group of ancient proteins that were ancestral to both thioredoxins and glutaredoxins.

An efficient route toward the synthesis of unsaturated (bis-diacetylenic) and saturated 40- and 48-membered macrocyclic biphosphocholines has been developed using 2-phenyl-5-hydroxy-1,3-dioxane as a common glycerol synthon. Ring closure was accomplished using either high-dilution Glaser oxidation of [(Cy3P)2RU==CHPh]Cl2-catalyzed olefin metathesis conditions. Deprotection of benzyl ethers using trimethylsilyl iodide (TMS-I) in the presence of diacetylenic moieties has also been demonstrated for the first time.

The contributions to thermostability of interactions within the beta-sheet region of rubredoxins (Rds) were investigated by examining proteins in which beta-strand sequences of Rds from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) and the mesophilic bacterium Clostridium pasteurianum (Cp) were interchanged. The thermostabilities of the chimeric Rds were assessed by monitoring the decay of the visible absorbance at 490 nm and of the far-UV CD vs time at 92 degrees C. The chimeric Rds Pf15 Cp47 Pf (Pf Rd residues 2-15 and 48-54 and Cp Rd residues 16-47) and Cp15 Pf47 Cp were both found to be far less thermostable than wild-type Pf Rd, indicating that neither the beta-sheet residues (2-7, 10-15, and 48-53) nor the "core residues" (16-47) of Pf Rd independently confer Pf Rd-like thermostability. However, the chimeric Rd Pf47 Cp exhibits thermostability close to that of wild-type Pf Rd, suggesting that Pf Rd-like thermostability is conferred by interactions of beta-sheet strands 1 and 2 (residues 2-15) together with Pf core residues. In contrast, Cp Rd beta-sheet strands 1 and 2 connecting to Pf Rd core residues are thermodestabilizing in the chimera Cp15 Pf Rd. These results suggest that a global alignment which optimizes both main chain and side chain interactions between beta-sheet strands and core residues is more important than a few localized interactions within the beta-sheet in conferring Pf Rd-like thermostability.

Molecular dynamics simulations are used to evaluate the temperature dependent differences in structure, solvation, and energies for the iron-sulfur protein rubredoxin from the hyperthermophilic archebacterium Pyrococcus furiosus to understand the unusual temperature dependence of its redox potential [Adams, M. W. W. (1992) Adv. Inorg. Chem. 38, 341-396]. Simulations of both redox states performed at 295 and 363 K reveal that almost no backbone structure alteration occurs at the higher temperature and that the radius of gyration of the protein is temperature and redox state independent. The most striking change is that the penetration of the redox site by solvent molecules in the reduced from at 295 K, which was also seen in simulations of the reduced form of the mesophilic Clostridium pasteurianum rubredoxin at 295 K (Yelle, R. B., et al. (1995) Proteins 22, 154-167], is no longer seen to a significant extent in either redox state at 363 K. Comparing 295 to 363 K, the calculated change in the electrostatic potential of about -300 mV and in the negative of the potential energy of about -550 meV is consistent with the observed change in redox potential of -160 mV. Moreover, the calculated change is in the wrong direction if the penetrating water is excluded. These results show that changing solvent accessibility may be responsible for the temperature dependence of the redox potential of P. furiosus rubredoxin.

A molecular model for the three-dimensional solution structure of the paramagnetic, four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been constructed on the basis of the reported 1H NMR spectral parameters [Donaire, A. (1996) J. Biomol. NMR 7, 35-47]. The conventional use of long mixing time NOESY cross-peak intensity, backbone angles, and hydrogenbonding constraints for building the structure was augmented by short mixing time NOESY, steady-state NOE, paramagnetic relaxation constraints, and the angular dependence of the ligated Cys H beta contact shifts. Distance geometry was used to generate various initial structures, and these structures were refined with the simulated annealing protocol. The family of structures with inconsequential violations exhibited low RMS deviations for the backbone except for a few residues in the immediate cluster vicinity and traces out a secondary structure very similar to those of the structurally characterized single cubane cluster Fds. The ability to describe the cluster environment depended on the use of numerous paramagnetic relaxation constraints which resulted in even the cluster loop residues exhibiting well-defined orientations, with the exception of one residue (Ilel1) whose 1H signals have not been located. Comparison of the structure of Tl Fd to those of mesophilic ferredoxins reveals that Tl Fd possesses the same secondary structural elements, two beta-sheets, two helices, and four turns, with the exception that the beta-sheet involving the termini incorporates a third strand in Tl Fd. Several minor structural adjustments in Tl Fd relative to other Fds, in addition to the third strand for beta-sheet, include the incorporation of the termini into the beta-sheet, a likely salt bridge from the side chain of the third beta-strand to the N-terminus, and a more hydrophobic and compact interaction between the large beta-sheet and the long helix. It is likely that each of these modifications, among others not yet well-defined (i.e., surface salt bridges), contributes to the extraordinary thermostability of Tl Fd.

The molecular and electronic structure of the four-iron cluster of the ferredoxin (Fd) from the hyperthermophilic archaeon, Pyrococcus furiosus, Pf (which has only three Cys in the cluster binding consensus sequence), has been investigated by 1H NMR in order to determine the identity of the noncysteinyl cluster ligand in each of the four redox states [Gorst, C. M., Zhou, Z. H., Ma, K., Teng, Q., Howard, J. B., Adams, M. W., & La Mar, G. N. (1995) Biochemistry 34, 8788-8795], and to characterize the electron spin ground state for the reduced cluster which at 10 K exhibits an unusual predominant S = 3/2 ground state [Conover, R. C., Kowal, A. T., Fu, W., Park, J. -B., Aono, S., Adams, M. W. W., & Johnson, M. K. (1990) J. Biol. Chem. 265, 8533-8541]. It is demonstrated that a combination of 1D and 2D NMR tailored to relaxed resonances allows the location of four hyperfine shifted and paramagnetically relaxed spin systems which dictates that all four cluster ligands are amino acid side chains, rather than a solvent water/hydroxide at the unique non-Cys ligation site. Three of the ligands could be sequence-specifically assigned to the three Cys residues (positions 11, 17, and 56) in the consensus sequence for cluster binding, hence identifying the fourth ligand as Asp 14. It is concluded that the identification of Asp ligation to a 4Fe cluster is readily achieved in the reduced, but not in the oxidized cluster of Fd. Analysis of the relaxation properties and pattern of the hyperfine shifts in Pf Fd reveals very strong similarities to other Fds with S = 1/2 ground states, leading to the conclusion that the S = 3/2 ground state is not detected in solution at ambient temperatures, and this in independent of the redox state of the two remaining Cys residues in the protein (positions 21 and 48). However, the electron self-exchange rate for 4Fe Pf Fd is significantly slower than for other 4Fe Fd with complete Cys ligation. Changes in the pattern of hyperfine shifts between oxidized and reduced clusters for the four ligands in Pf Fd reveal that the most significant variation occurs for the Asp 14 orientation, suggesting that the altered Asp orientation may "gate" the electron transfer.

The bases of the hyperthermostability of rubredoxin from Pyrococcus furiosus (RdPf) have been probed by structural perturbations induced by solution pH and ionic strength changes. Comparison of the solution behavior at pH 7 and pH 2, as probed by far- and near-UV circular dichroism, Trp fluorescence emission, 1-anilinonaphthalene-8-sulfonate (ANS) binding, and NMR spectroscopy, reveals the presence of only minimal structural variations at room temperature. At pH 2, the protein displays a surprising nearly native-like behavior at high ionic strength while, at low ionic strength, it is capable of strongly binding the hydrophobic probe ANS. All the secondary and tertiary structural features, including the environment of the hydrophobic core, appear to be intact regardless of pH and ionic strength. The apparent "melting" or denaturation temperature at pH 2, however, is 42 degrees C lower than at pH 7. This is attributed to the perturbation of many electrostatic interactions, including the disruption of all the ion pairs, which is complete at pH 2, as indicated by electrometric pH titrations. The implications of these findings for the origins of the hyperthermostability of rubredoxin are discussed.

The oxidized and reduced forms of the [4Fe-4S]-containing ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, Pf, have been investigated by 1H nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and thiol titrations. We have identified and isolated at Ambient temperature four distinct redox states for the [4Fe-4S] form of the ferredoxin. These states differ in the redox state of the cluster, which is coordinated by Cys 11, Asp 14, Cys 17, and Cys 56, and of a disulfide bridge between Cys 21 and Cys 48. The protein, as isolated under anaerobic conditions, designated 4Fe FdBred, contains the reduced cluster and two free thiols. The cluster, but not the thiols, is readily oxidized by brief exposure to O2 to yield 4Fe FdBOX. Prolonged O2 treatment (> 24 h at 30 degrees C) is required to generate the protein with a disulfide (4Fe FdAOX) while this fully oxidized form is readily converted by brief reduction with sodium dithionite to the protein with a reduced cluster and a disulfide (4Fe FdAred). Analyses of the magnitude and the number of hyperfine-shifted resonances in each of the four redox states are discussed.

One- and two-dimensional 1H NMR data tailored to detect paramagnetically relaxed protons near the S = 1/2, three-iron-sulfur cluster of the ferredoxin from the hyperthermophile Pyrococcus furiosus are analyzed to sequence specifically assign the hyperfine shifted ligated cysteine signals, to determine the nature of the secondary structural elements on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterized secondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse, S.C., Howard, J.B., Adams, M. W. W., & La Mar, G. N. (1994) Biochemistry 33, 6316-6326]. Inspection of the geometry of the cluster ligating cysteines in the six structurally characterized cubane ferredoxin (Fd) clusters reveals a pattern of distances from the cluster iron(s) that indicate that each Cys will exhibit one backbone proton that will allow the detection of dipolar connectivities to the backbone of adjacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibit weakly relaxed peptide NH and strongly relaxed C alpha H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weakly relaxed C alpha H peaks. These dipolar contacts are clearly observed for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explicitly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferredoxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys17 is on a short helix through Leu20 and that Cys56 likely initiates a type I turn, as observed in the crystal structure of the 3Fe Fd for Desulfovibrio gigas [Kissinger, C. R., Sieker, L. C., Adman, E. T., & Jensen, L. H. (1991) J. Mol. Biol. 219, 693-715]. The observed relaxation rates of resolved or partially resolved signals are shown to correlate with their proximity to the various iron in the cluster, as determined for the homologous residues in D. gigas Fd, providing additional qualitative information on tertiary contacts of the cluster.

The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771]. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711-5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.

Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Halocyanin is a recently discovered archaebacterial copper protein classified as "type I" small blue copper protein (Scharf, B., Ph.D. Thesis, University of Bochum, Germany). Its redox properties were investigated by a combination of protein electrochemical and spectroscopic techniques. Using electrochemical reactions in an ultrathin-layer electrochemical cell developed for UV/vis and IR spectroscopy, halocyanin could be quantitatively and reversibly oxidized and reduced. The titration of the absorption band at 600 nm can be perfectly described by a Nernst curve with n = 1 electron transferred; a quantitative fit yields a midpoint potential, Em, of 183 mV (vs SHE) at a pH of 7.3. The midpoint potential falls constantly from +333 mV at pH 4 to +119 mV at pH 10, with three regions around pH 4.5, 6.5, and 8.5 where the pH dependence is ca. -60 mV/pH unit, indicating the uptake of a proton with the reduction. By analogy with other small type I copper proteins, the three pK values suggested by the pH dependency of Em might be associated with three histidines which interact with the redox site. Electrochemically induced reduced-minus-oxidized Fourier transform infrared difference spectra in the 1800-1000 cm-1 range at neutral pH show a number of strong difference bands between ca. 1700 and 1600 cm-1 as well as smaller difference structures between 1600 and 1200 cm-1. The maximum amplitude of the difference bands--only ca. 1% of the amide-I absorption at ca. 1639 cm-1--indicates that only small protein rearrangements occur upon the redox transition.(ABSTRACT TRUNCATED AT 250 WORDS)

50S ribosomal subunits from the extreme halophilic archaebacterium Haloarcula marismortui were treated with the homobifunctional protein-protein cross-linking reagents diepoxybutane (4 A) and dithiobis(succinimidyl propionate) (12 A). The dominant product with both cross-linking reagents was identified on the protein level as HmaL23-HmaL29, which is homologous to the protein pair L23-L29 from Escherichia coli [Walleczek, J., Martin, T., Redl, B., Stöffler-Meilicke, M., & Stöffler, G. (1989) Biochemistry 28, 4099-4105] and from Bacillus stearothermophilus [Brockmöller, J., & Kamp, R. M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935]. To reveal the exact cross-linking site in HmaL23-HmaL29, the cross-linked complex was purified on a preparative scale by conventional and high-performance liquid chromatography. After endoproteolytic fragmentation of the protein pair, the amino acids engaged in cross-link formation were unambiguously identified by N-terminal sequence analysis and mass spectrometry of the cross-linked peptides. The cross-link is formed between lysine-57 in the C-terminal region of HmaL29 and the alpha-amino group of the N-terminal serine in protein HmaL23, irrespective of the cross-linking reagent. This result demonstrates that the N-terminal region of protein HmaL23 and the C-terminal domain of HmaL29 are highly flexible so that the distance between the two polypeptide chains can vary by at least 8 A. Comparison of our cross-linking results with those obtained with B. stearothermophilus revealed that the fine structure within this ribosomal domain is at least partially conserved.

The structures of the modified folates present in Pyrococcus furiosus have been determined. This was accomplished largely by the characterization of the arylamines resulting from the air oxidative cleavage of the reduced modified folates present in these cells, using both chemical and enzymatic methods. Cell extracts separated on DEAE-Sephadex columns showed one major peak containing the arylamines derived from the modified folates. These arylamines were not retained on the DEAE-Sephadex columns, indicating that they contained no net negative charge. Purification of the azo dye derivatives of these arylamines on a Bio-Gel P-6 column showed the presence of three different compounds (compounds 1, 2, and 3) in an average amount of 4.1, 7.6, and 22 nmol/g dry weight of cells, respectively. Each of these compounds readily underwent mild acid hydrolysis (0.1 M HCl, 110 degrees C, 1 min) to produce the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane (pAPT). The structure and stereochemistry (ribo) of the pAPT was the same as the pAPT present in methanopterin. In addition, compounds 1, 2, and 3 were each shown to contain 1 mol equiv of ribose and 1, 2, and 3 mol equiv of N-acetylglucosamine (gluNAc), respectively, and were designated as the azo dye derivatives of pAPT-ribose-gluNAc, pAPT-ribose-(gluNAc)2, and pAPT-ribose-(gluNAc)3. Each of these compounds was readily cleaved to the azo dye derivative of pAPT-ribose by the enzymatic action of beta-N-acetylglucosaminidase, indicating that all the gluNAc residues were beta-linked.(ABSTRACT TRUNCATED AT 250 WORDS)

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.