Surface pressure-dependent interactions of secretory phospholipase A2 with zwitterionic phospholipid membranes

The hydrolytic activity of secretory phospholipase A(2) (PLA(2)) is regulated by many factors, including the physical state of substrate aggregates and the chemical nature of phospholipid molecules. In order to achieve strong binding of PLA(2) on its substrates, many previous works have used anionic lipid dispersion to characterize the orientation and penetration depth of PLA(2) molecules on membrane surfaces. In this study, we applied monolayer technique with controllable surface area to investigate the PLA(2)s of Taiwan cobra venom and bee venom on zwitterionic phophatidylcholine monolayers and demonstrated an optimum hydrolytic activity at a surface pressure of 18 and 24 mN/m, respectively. By combining polarized attenuated total reflection Fourier-transform infrared spectroscopy and monolayer-binding experiments, we found that the amount of membrane-bound PLA(2) decreased markedly as the surface pressure of the monolayer was increased. Interestingly, the insertion area of the PLA(2)s decreased to near zero as the surface pressure increased to the optimum pressure for hydrolytic activity. On the basis of the measured infrared dichroic ratio, the orientation of the PLA(2)s bound to zwitterionic membranes was similar to that observed on a negatively charged membrane and was independent of the surface pressure. Our findings suggest that both PLA(2)s were located on the membrane surface rather than penetrating the membrane bilayer and that the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes. The results are discussed in terms of the easy access of catalytic water for the PLA(2) activity and the mobilization of its substrate and product to facilitate the catalytic process.     

Surface-dependent transitions during self-assembly of phospholipid membranes on mica, silica, and glass

Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication. Results were compared with membrane formation on silica and glass, and the influence of stirring, buffer, and calcium was assessed. Without calcium, DOPC vesicles had a low affinity (Kd approximately 30 microM) for mica, and DOPS/DOPC vesicles hardly adsorbed. Addition of calcium promptly caused condensation of the adhering vesicles, with either loss of excess lipid or rapid additional lipid adsorption up to full surface coverage. Vesicle-mica interactions dominate the adsorption process, but vesicle-vesicle interactions also seem to be required for the condensation process. Membranes on mica proved unstable in Tris-HCl buffer. For glass, transport-limited adsorption of DOPC and DOPS/DOPC vesicles with immediate condensation into bilayers was observed, with and without calcium. For silica, vesicle adsorption was also rapid, even in the absence of calcium, but the transition to condensed layers required a critical surface coverage of about 50% of bilayer mass, indicating vesicle-vesicle interaction. For all three surfaces, additional adsorption of DOPC (but not DOPS/DOPC) vesicles to condensed membranes was observed. DOPC membranes on mica were rapidly degraded by phospholipase A2 (PLA2), which pleads against the role of membrane defects as initial PLA2 targets. During degradation, layer thickness remained unchanged while layer density decreased, in accordance with recent atomic force microscopy measurements of gel-phase phospholipid degradation by PLA2.     

Selection of hydrophobic membranes in the lipase-catalyzed hydrolysis of olive oil

A hydrophobic membrane (HVHP, polyvinylidene difluoride) was selected out of HVHP, PTHK and PTGC (polysulfone) membranes to immobilize Candida rugosa lipase by physical adsorption in the hydrolysis of olive oil in a stirred diffusion cell. A previous model that assumed the Michaelis–Menten kinetics and Langmuir adsorption isotherm for the adsorbed lipase was used to interpret the variation of initial hydrolysis rates with enzyme and substrate concentrations. Replacing the aqueous phase by a fresh buffer, with or without containing partially deactivated lipases, during the reaction did not affect the enzyme activity for the adsorbed lipase. Moreover, the same enzyme performance was obtained when a fresh and a regenerated membrane was used as the carrier in the membrane reactor.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles

We have investigated the adsorption of phospholipid mixtures using neutron reflection. Small sonicated unilamellar vesicles (SUV) composed of DOPC and d(62)-DPPC were incubated at 50 degrees C in contact with a silica surface using a method commonly employed to form supported model membranes. The composition of the mixed supported bilayer was found to be substantially different from that of the bulk vesicles in a direction indicating a higher affinity of DPPC for the silica surface. Formation of an asymmetric bilayer arrangement was also discovered in all the cases studied. DPPC tended to dominate the composition of the leaflet next to silica, while the outer leaflet was generally closer to the bulk composition. The supported bilayers also exhibited increasing interfacial roughness in the outer membrane leaflet in the region of the DOPC-DPPC gel-liquid immiscibility region. To our knowledge, this is the first time that both the structure and the absolute composition of a mixed-lipid supported bilayer have been resolved, and the results raise a number of questions regarding the adsorption of vesicles and the properties of supported bilayers, which are discussed in terms of the bulk phase diagram of DOPC and DPPC.     

Surface enzyme kinetics for biopolymer microarrays: a combination of Langmuir and Michaelis-Menten concepts

Real-time surface plasmon resonance (SPR) imaging measurements of surface enzymatic reactions on DNA microarrays are analyzed using a kinetics model that couples the contributions of both enzyme adsorption and surface enzyme reaction kinetics. For the case of a 1:1 binding of an enzyme molecule (E) to a surface-immobilized substrate (S), the overall enzymatic reaction can be described in terms of classical Langmuir adsorption and Michaelis-Menten concepts and three rate constants: enzyme adsorption (k(a)), enzyme desorption (k(d)) and enzyme catalysis (k(cat)). In contrast to solution enzyme kinetics, the amount of enzyme in solution is in excess as compared to the amount of substrate on the surface. Moreover, the surface concentration of the intermediary enzyme-substrate complex (ES) is not constant with time, but goes to zero as the reaction is completed. However, kinetic simulations show that the fractional surface coverage of ES on the remaining unreacted sites does reach a steady-state value throughout the course of the surface reaction. This steady-state value approaches the Langmuir equilibrium value for cases where k(a)[E] > k(cat). Experiments using the 3' --> 5' exodeoxyribonuclease activity of Exonuclease III on double-stranded DNA microarrays as a function of temperature and enzyme concentration are used to demonstrate how this model can be applied to quantitatively analyze the SPR imaging data.     

Surface pressure dependence of phospholipase A2 activity in lipid monolayers is linked to interfacial water activity

The specific activity of pancreatic phospholipase A2 (PLA2) was studied in two disparate systems, one involving phosphatidylcholine monolayer at various surface pressures at the air-water interface and the other involving a solid-state system exposed to various equilibrium relative humidity (ERH). The results were examined in terms of thermodynamic activity of water in the interfacial region (aws*) and in the hydrated solid phase (aw). In both these physically different systems, the specific activity versus aw and aws* profiles of PLA2 were remarkably similar. In both cases, the specific activity exhibited a maximum at aw (or aws*) approximately 0.3. These results suggested that the mechanism of control of PLA2 activity at the lipid-water interface might involve modulation of the hydration state of the enzyme through control of the thermodynamic activity of water in the interfacial region. Extension of these results to biomembranes suggests that one of the functions of lipid bilayer might be the control of local water activity at the lipid-water interface. In biological membranes, localized subtle changes in interfacial water activity may occur as a result of local stretching or compression of the membrane facilitated by conformational changes in membrane-bound receptor proteins.     

充蜡石墨支撑的季铵离子/己二酸混合双层膜上钙离子诱发的离子通道行为

以充蜡石墨电极作为新型支撑体,成功制备了一种季铵离子为内层（包括四丁 基铵TBA,十六烷基三甲基铵CTrMA）,己二酸（HDA）为外层的新型自组装混合双 层膜,以循环伏安和电化学交流阻抗方法研究了膜的离子通道行为。该膜能够接受 Ca~(2+)的刺激作用而打开[Fe(CN)_6]~(3-/4-)电极氧化还原的离子通道,撤走该 刺激离子则通道关闭。提出了混合双层膜的结构和离子通道作用的模型,指出外层 膜HDA分子可能具有V型和W型两种结构。     

Growth of giant membrane lobes mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces

We report on the growth of giant membrane lobes that is mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces and a strategy to control the two-dimensional structure of the membrane lobes on a solid surface. The growth of giant membrane lobes was observed on a single-lipid bilayer which spread from a lump of phospholipid deposited on a silica-glass substrate or an oxidized silicon wafer in aqueous solutions of NaCl, KCl, MgCl2, or CaCl2 at relatively high salt concentrations. Most of the membrane lobes were very flat unilamellar tubes elongating from the lump of phospholipid, and their length reached 1 mm in 5 h. Experimental findings clearly indicate that the membrane lobes are adherent to the surface of the single-lipid bilayer and are mechanically elongated from the lump of phospholipid by the sliding motion of the single-lipid bilayer. We could control the two-dimensional structure of the membrane lobes on the substrate by controlling the spreading direction of the single-lipid bilayer using Pt micropatterns that were deposited on the smooth surface of the oxidized silicon wafer.     

Oxide-dependent adsorption of a model membrane phospholipid, dipalmitoylphosphatidylcholine: bulk adsorption isotherms

The importance of substrate chemistry and structure on supported phospholipid bilayer design and functionality is only recently being recognized. Our goal is to investigate systematically the substrate-dependence of phospholipid adsorption with an emphasis on oxide surface chemistry and to determine the dominant controlling forces. We obtained bulk adsorption isotherms at 55 degrees C for dipalmitoylphosphatidylcholine (DPPC) at pH values of 5.0, 7.2, and 9.0 and at two ionic strengths with and without Ca(2+), on quartz (alpha-SiO(2)), rutile (alpha-TiO(2)), and corundum (alpha-Al(2)O(3)), which represent a wide a range of points of zero charge (PZC). Adsorption was strongly oxide- and pH-dependent. At pH 5.0, adsorption increased as quartz < rutile approximately corundum, while at pH 7.2 and 9.0, the trend was quartz approximately rutile < corundum. Adsorption decreased with increasing pH (increasing negative surface charge), although adsorption occurred even at pH > or = PZC of the oxides. These trends indicate that adsorption is controlled by attractive van der Waals forces and further modified by electrostatic interactions of oxide surface sites with the negatively charged phosphate ester (-R(PO(4)-)R'-) portion of the DPPC headgroup. Also, the maximum observed adsorption on negatively charged oxide surfaces corresponded to roughly two bilayers, whereas significantly higher adsorption of up to four bilayers occurred on positively charged surfaces. Calcium ions promote adsorption beyond a second bilayer, regardless of the sign of oxide surface charge. We develop a conceptual model for the structure of the electric double layer to explain these observations.     

Removal of RNA during protein concentration by means of enzyme membrane

Immobilization of RNase in PVC ultrafiltration membranes was carried out. The obtained membranes were used for concentration of BSA solution, RNA being simultaneously removed. The yield of RNA hydrolysis was found to be controlled by the initial concentration of RNA in feed solution. The protein affected enzyme action as a result of its adsorption on the membrane surface at the beginning of ultrafiltration, whereas it did not inhibit RNase activity during the process.     

Structuring of supported hybrid phospholipid bilayers on electrodes with phospholipase A2

The effect of a lipolytic enzyme, pork pancreatic phospholipase A(2), on hybrid bilayer membranes was monitored using voltammetry, impedance spectroscopy and surface plasmon resonance. The hybrid bilayers were prepared by Langmuir-Schaefer transfer of lipid monolayers onto gold electrodes modified with self-assembled alkanethiol monolayers, or by liposome spreading. The electrodes were immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and cleavage of the ester bond in the sn-2 position of phospholipids in the outer leaflet of the hybrid layers. The action of phospholipase A(2) led to perforation of the lipid films. Impedance spectroscopy and surface plasmon resonance were used for monitoring enzyme adsorption, phospholipid hydrolysis and product desorption. The results obtained show that transport efficiency of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the bilayer depends on the action of the enzyme; the state of the lipid layer covering the electrode surface depends on the latter as well. Cyclic voltammetry and electrochemical impedance spectroscopy were used to study this effect. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using a bare gold electrode indicated facilitated penetration of the drug into the layer. The results obtained were interpreted in terms of pore formation in the lipid matrix; phospholipase A(2) can be considered as a nano-device for high precision perforation of the lipid layer.     

Adsorption dynamics of water on Pt{110}-(1 x 2)

The dynamics of H(2)O adsorption on Pt{110}-(1 x 2) is studied using supersonic molecular beam and temperature programed desorption techniques. The sticking probabilities are measured using the King and Wells method at a surface temperature of 165 K. The absolute initial sticking probability s(0) of H(2)O is 0.54+/-0.03 for an incident kinetic energy of 27 kJmol. However, an unusual molecular beam flux dependence on s(0) is also found. At low water coverage (theta<1), the sticking probability is independent of coverage due either to diffusion in an extrinsic precursor state formed above bilayer islands or to incorporation into the islands. We define theta=1 as the water coverage when the dissociative sticking probability of D(2) on a surface predosed with water has dropped to zero. The slow falling H(2)O sticking probability at theta>1 results from compression of the bilayer and the formation of multilayers. Temperature programed desorption of water shows fractional order kinetics consistent with hydrogen-bonded islands on the surface. A remarkable dependence of the initial sticking probability on the translational (1-27 kJ/mol) and internal energies of water is observed: s(0) is found to be essentially a step function of translational energy, increasing fivefold at a threshold energy of 5 kJ/mol. The threshold migrates to higher energies with increasing nozzle temperature (300-700 K). We conclude that both rotational state and rotational alignment of the water molecules in the seeded supersonic expansion are implicated in dictating the adsorption process.     

Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed 15N NMR relaxation

Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR (15)N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein-membrane interactions.     

Enzymatic hydrolysis by cutinase of PEG-co PLA copolymers spread monolayers

The mechanism of the hydrolysis by cutinase and the progressive fragmentation of lactic chains in diblock copolymers of PLA with various sizes attached to PEG were studied in a 2D monolayers model system. The hydrolysis kinetics was followed by measuring simultaneously the decrease of the surface area and evolution of the surface potential with time at barostatic conditions. The decrease of the surface area is due to the solubilization of the copolymers as well as of their hydrolytic products: detached PEG blocks and small soluble PLA fragments. The evolution of the surface potential detects the transient interfacial accumulation of charged insoluble PLA fragments. A kinetic model describing the enzymatic hydrolysis was developed and the values for the global hydrolytic kinetic constant were obtained without any fitting parameter. It was found that the global kinetic constant no practically depend on the length of the lactic and the presence of polyethylene–glycol chains.     

Kinetics of adsorption and proteolytic cleavage of a multilayer ovalbumin film by subtilisin Carlsberg

Adsorption and proteolytic activity of the enzyme subtilisin Carlsberg have been studied on an immobilized, multilayer ovalbumin film. The cross-linked multilayer substrate permits protease adsorption to be examined unencumbered by the surface inhomogeneity typically observed in monolayer studies of protease surface kinetics. Decline of the protein film was measured over time using ellipsometry. Resulting kinetic data as a function of aqueous enzyme concentration and temperature were well fit by a Langmuir-Michaelis-Menten model for surface proteolysis. We observed that both the protein degradation kinetics and the in situ adsorption data were well described by the proposed model. The temperature dependence of the kinetic rate parameter yielded an activation energy of 12 kcal/mol. Further, the apparent Langmuir adsorption equilibrium constant of the enzyme at the protein/aqueous interface was 0.11 L/mg at 22 degrees C, 0.034 L/mg at 36 degrees C, and 0.011 L/mg at 50 degrees C. Although enzyme adsorption at a given aqueous enzyme concentration decreased at higher temperature, the enzyme cleaved the substrate more rapidly, leading to a net increase in the ovalbumin film degradation rate. We observed that the maximum enzyme coverage on the immobilized protein surface was approximately 40% of a close-packed monolayer at ambient temperature (22 degrees C).     

Lipid bilayer membrane with atomic step structure: supported bilayer on a step-and-terrace TiO2(100) surface

The formation of a supported planar lipid bilayer (SPLB) and its morphology on step-and-terrace rutile TiO 2(100) surfaces were investigated by fluorescence microscopy and atomic force microscopy. The TiO 2(100) surfaces consisting of atomic steps and flat terraces were formed on a rutile TiO 2 single-crystal wafer by a wet treatment and annealing under a flow of oxygen. An intact vesicular layer formed on the TiO 2(100) surface when the surface was incubated in a sonicated vesicle suspension under the condition that a full-coverage SPLB forms on SiO 2, as reported in previous studies. However, a full-coverage, continuous, fluid SPLB was obtained on the step-and-terrace TiO 2(100) depending on the lipid concentration, incubation time, and vesicle size. The SPLB on the TiO 2(100) also has step-and-terrace morphology following the substrate structure precisely even though the SPLB is in the fluid phase and an approximately 1-nm-thick water layer exists between the SPLB and the substrate. This membrane distortion on the atomic scale affects the phase-separation structure of a binary bilayer of micrometer order. The interaction energy calculated including DLVO and non-DLVO factors shows that a lipid membrane on the TiO 2(100) gains 20 times more energy than on SiO 2. This specifically strong attraction on TiO 2 makes the fluid SPLB precisely follow the substrate structure of angstrom order.     

Membrane orientation of MSI-78 measured by sum frequency generation vibrational spectroscopy

Antimicrobial peptides (AMPs) selectively disrupt bacterial cell membranes to kill bacteria whereas they either do not or weakly interact with mammalian cells. The orientations of AMPs in lipid bilayers mimicking bacterial and mammalian cell membranes are related to their antimicrobial activity and selectivity. To understand the role of AMP-lipid interactions in the functional properties of AMPs better, we determined the membrane orientation of an AMP (MSI-78 or pexiganan) in various model membranes using sum frequency generation (SFG) vibrational spectroscopy. A solid-supported single 1,2-dipalmitoyl-an-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) bilayer or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) bilayer was used as a model bacterial cell membrane. A supported 1,2-dipalmitoyl-an-glycero-3-phosphocholine (DPPC) bilayer or a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was used as a model mammalian cell membrane. Our SFG results indicate that the helical MSI-78 molecules are associated with the bilayer surface with ～70° deviation from the bilayer normal in the negatively charged gel-phase DPPG bilayer at 400 nM peptide concentration. However, when the concentration was increased to 600 nM, MSI-78 molecules changed their orientation to make a 25° tilt from the lipid bilayer normal whereas multiple orientations were observed for an even higher peptide concentration in agreement with toroidal-type pore formation as reported in a previous solid-state NMR study. In contrary, no interaction between MSI-78 and a zwitterionic DPPC bilayer was observed even at a much higher peptide concentration (～12,000 nM). These results demonstrate that SFG can provide insights into the antibacterial activity and selectivity of MSI-78. Interestingly, the peptide exhibits a concentration-dependent membrane orientation in the lamellar-phase POPG bilayer and was also found to induce toroidal-type pore formation. The deduced lipid flip-flop from SFG signals observed from lipids also supports MSI-78-induced toroidal-type pore formation.     

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.