High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection.

Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

On the Origins of Fracture Toughness in Advanced Teleosts: How the Swordfish Sword's Bone Structure and Composition Allow for Slashing under Water to Kill or Stun Prey.

The osseous sword of a swordfish (Xiphias gladius) is specialized to incapacitate prey with stunning blows. Considering the sword's growth and maturation pattern, aging from the sword's base to the tip, while missing a mechanosensitive osteocytic network, an in-depth understanding of its mechanical properties and bone quality is lacking. Microstructural, compositional, and nanomechanical characteristics of the bone along the sword are investigated to reveal structural mechanisms accounting for its exceptional mechanical competence. The degree of mineralization, homogeneity, and particle size increase from the base toward the tip, reflecting aging along its length. Fracture experiments reveal that crack-growth toughness vastly decreases at the highly and homogeneously mineralized tip, suggesting the importance of aging effects. Initiation toughness, however, is unchanged suggesting that aging effects on this hierarchical level are counteracted by constant mineral/fibril interaction. In conclusion, the sword of the swordfish provides an excellent model reflecting base-to-tip-wise aging of bone, as indicated by increasing mineralization and decreasing crack-growth toughness toward the tip. The hierarchical, structural, and compositional changes along the sword reflect peculiar prerequisites needed for resisting high mechanical loads. Further studies on advanced teleosts bone tissue may help to unravel structure-function relationships of heavily loaded skeletons lacking mechanosensing cells.

LabDisk for SAXS: a centrifugal microfluidic sample preparation platform for small-angle X-ray scattering.

We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 µl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7-4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines.

Enhancing actions of peptides derived from the γ-chain of fetal human hemoglobin on the immunostimulant activities of monophosphoryl lipid A.

Hemoglobin and its structures have been described since the 1990s to enhance a variety of biological activities of endotoxins (LPS) in a dose-dependent manner. To investigate the interaction processes in more detail, the system was extended by studying the interactions of newly designed peptides from the γ-chain of human hemoglobin with the adjuvant monophosphoryl lipid A (MPLA), a partial structure of lipid A lacking its 1-phosphate. It was found that some selected Hbg peptides, in particular two synthetic substructures designated Hbg32 and Hbg35, considerably increased the bioactivity of MPLA, which alone was only a weak activator of immune cells. These findings hold true for human mononuclar cells, monocytes and T lymphocytes. To understand the mechanisms of action in more detail, biophysical techniques were applied. These showed a peptide-induced change of the MPLA aggregate structure from multilamellar into a non-lamellar, probably inverted, cubic structure. Concomitantly, the peptides incorporated into the tightly packed MPLA aggregates into smaller units down to monomers. The fragmentation of the aggregates was an endothermic process, differing from a complex formation but rather typical for a catalytic reaction.

Biophysical analysis of the interaction of the serum protein human ß2GPI with bacterial lipopolysaccharide.

There are several human serum proteins for which no clear role is yet known. Among these is the abundant serum protein beta2-glycoprotein-I (ß2GPI), which is known to bind to negatively charged phospholipids as well as to bacterial lipopolysaccharides (LPS), and was therefore proposed to play a role in the immune response. To understand the details of these interactions, a biophysical analysis of the binding of ß2GPI to LPS and phosphatidylserine (PS) was performed. The data indicate only a moderate tendency of the protein (1) to influence the LPS-induced cytokine production in vitro, (2) to react exothermally with LPS in a non-saturable way, and (3) to change its local microenvironment upon LPS association. Additionally, we found that the protein binds more strongly to phosphatidylserine (PS) than to LPS. Furthermore, ß2GPI converts the LPS bilayer aggregates into a stronger multilamellar form, and reduces the fluidity of the hydrocarbon moiety of LPS due to a rigidification of the acyl chains. From these data it can be concluded that ß2GPI plays a role as an immune-modulating agent, but there is much less evidence for a role in immune defense against bacterial toxins such as LPS.

Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity.

The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 µm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.

Biophysical mechanisms of the neutralization of endotoxins by lipopolyamines.

Endotoxins (lipopolysaccharides, LPS) are one of the strongest immunostimulators in nature, responsible for beneficial effects at low, and pathophysiological effects at high concentrations, the latter frequently leading to sepsis and septic shock associated with high mortality in critical care settings. There are no drugs specifically targeting the pathophysiology of sepsis, and new therapeutic agents are therefore urgently needed. The lipopolyamines are a novel class of small molecules designed to sequester and neutralize LPS. To understand the mechanisms underlying the binding and neutralization of LPS toxicity, we have performed detailed biophysical analyses of the interactions of LPS with candidate lipopolyamines which differ in their potencies of LPS neutralization. We examined gel-to-liquid crystalline phase behavior of LPS and of its supramolecular aggregate structures in the absence and presence of lipopolyamines, the ability of such compounds to incorporate into different membrane systems, and the thermodynamics of the LPS:lipopolyamine binding. We have found that the mechanisms which govern the inactivation process of LPS obey similar rules as found for other active endotoxin neutralizers such as certain antimicrobial peptides.

Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus.

Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H(1) and H(2) from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H(1) and H(2) with astaxanthin reproduced the bathochromic shift of 85-95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype-phenotype linkage.

Biophysical investigations into the interactions of endotoxins with bile acids.

The interaction of selected endotoxin preparations (lipid A from Erwinia carotovora and LPS Re and Ra from Salmonella enterica sv. Minnesota strains R595 and R60, respectively) with selected bile acids was investigated biophysically. Endotoxin aggregates were analyzed for their gel-to-liquid crystalline phase behavior, the type of their aggregates, the conformation of particular functional groups, and their Zeta potential in the absence and presence of the bile acids by applying Fourier-transform infrared spectroscopy, differential scanning calorimetry, measurements of the electrophoretic mobility, and synchrotron radiation X-ray scattering. In addition, the ability of the endotoxins to induce cytokines in human mononuclear cells was tested in the absence and presence of varying concentrations of bile acids. The data show that the endotoxin:bile acid interaction is not governed by Coulomb forces, rather a hydrophobic interaction takes place. This leads to an enhanced formation of the inherent cubic aggregate structures of the endotoxins, concomitant with a slight disaggregation, as evidenced by freeze-fracture electron microscopy. Parallel to this, the addition of bile acids increased the bioactivity of lipid A and, to a lower degree, also that of the tested rough mutant LPS at lower concentrations of the endotoxin preparation, a finding similar as reported for the interaction of other agents such as hemoglobin. These data imply that there are general mechanisms that govern the expression of biological activities of endotoxins.

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.

New antiseptic peptides to protect against endotoxin-mediated shock.

Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.

Crystal and solution structure of the C-terminal part of the Methanocaldococcus jannaschii A1AO ATP synthase subunit E revealed by X-ray diffraction and small-angle X-ray scattering.

The structure of the C-terminus of subunit E (E(101-206)) of Methanocaldococcus jannaschii A-ATP synthase was determined at 4.1 A. E(101-206) consist of a N-terminal globular domain with three alpha-helices and four antiparallel beta-strands and an alpha-helix at the very C-terminus. Comparison of M. jannaschii E(101-206) with the C-terminus E(81-198) subunit E from Pyrococcus horikoshii OT3 revealed that the kink in the C-terminal alpha-helix of E(81-198), involved in dimer formation, is absent in M. jannaschii E(101-206). Whereas a major dimeric surface interface is present between the P. horikoshii E(81-198) molecules in the asymmetric unit, no such interaction could be found in the M. jannaschii E(101-206) molecules. To verify the oligomeric behaviour, the low resolution structure of the recombinant E(85-206) from M. jannaschii was determined using small angle X-ray scattering. Rigid body modeling of two copies of one of the monomer established a fit with a tail to tail arrangement.

Golgi protein FAPP2 tubulates membranes.

The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network.

A different conformation for EGC stator subcomplex in solution and in the assembled yeast V-ATPase: possible implications for regulatory disassembly.

Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy. In solution, EG forms a slightly kinked rod, which assembles with subunit C into an L-shaped structure. This model is supported by the microscopy data, which show three copies of EG with two of these linked by subunit C. However, the relative arrangement of the EG and C subunits in solution is more open than that in the holoenzyme, suggesting a conformational change of EGC during regulatory assembly and disassembly.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Physico-chemical and biophysical study of the interaction of hexa- and heptaacyl lipid A from Erwinia carotovora with magainin 2-derived antimicrobial peptides.

The neutralization of endotoxin structures such as the active 'endotoxic principle' lipid A by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria which frequently may lead to the septic shock syndrome. An effective antimicrobial peptide, originally found in the skin of an African frog, is magainin 2. Here, the interaction of magainin 2-amide and a peptide derived thereof, M2V, with chemically defined and homogeneous hexaacyl and heptaacyl lipids A isolated from LPS of Erwinia carotovora, was investigated. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A and the conformation of their phosphate groups due to peptide binding was investigated. The former parameter was also determined by using differential scanning calorimetry. The electrophoretic mobility of lipid A aggregates under the influence of the peptides was studied to determine the Zeta potential, and small-angle X-ray scattering was applied for the elucidation of the types of aggregate structures in the absence and presence of the peptides. The lipid A-induced cytokine production in human mononuclear cells shows that the ability of the two peptides to inhibit a tumor necrosis factor-alpha production correlates with characteristic changes of the biophysical parameters. These are much stronger expressed for the peptide M2V than for magainin 2-amide, which apparently is connected with the higher number of positive as well as more hydrophobic amino acids, leading to a stronger amphiphilicity necessary to neutralize the amphiphilic lipid A aggregates.

Structural polymorphism of hydrated ether-linked dimyristyl maltoside and melibioside.

The structural polymorphism of two selected disaccharide glycolipids with a maltose (DMMA) and a melibiose (DMME) carbohydrate headgroup linked to dimyristyl alkyl chains were investigated by FTIR-spectroscopy, differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and film-balance measurements. The compounds displayed thermotropic multilamellar phases. In the gel phase, DMMA formed also a crystalline phase of orthorhombic symmetry, and DMME an interdigitated phase. The gel to liquid crystalline phase transition temperature T(c) of DMMA depended on the storage and hydration conditions, a precooled sample having a T(c) around 45 degrees C, and a freshly prepared sample around 33 degrees C. In contrast, the phase transition temperature for the gel to liquid crystalline phase of DMME was always found at 24 degrees C. Surface pressure isotherms of the lipids on water and buffer showed that DMMA covers only a small surface area (approximately 35A(2)) whereas DMME requires 50 A(2) of space on the surface. Films of DMMA can be compressed up to a maximum compressibility Pi(max) of 54 mN m(-1) whereas the tilted DMME forms less stable films with Pi(max) of 34 mN m(-1). These different structural characteristics reflect the different conformations of the disaccharide head groups. The presence of the alpha1-->4 linked maltose head group in DMMA and an alpha1-->6 linked melibiose head group in DMME induces geometrical structures ranging from a slightly wedge-shaped towards a more tilted structure, and as a consequence of Israelachvilis packing model, to the formation of different phases. In addition, the structural constraints of DMME allow the formation of a phase with interdigitated hydrocarbon chains.

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins.

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.

Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamic, biophysical and microbiological analysis.

On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.

Physicochemical and biological analysis of synthetic bacterial lipopeptides: validity of the concept of endotoxic conformation.

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle.