Nano-scale study of the nucleation and growth of calcium phosphate coating on titanium implants.

The nucleation and growth of a calcium phosphate (Ca-P) coating deposited on titanium implants from simulated body fluid was investigated by using atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Forty titanium alloy plates were assigned into two groups. One group with a smooth surface having a maximum roughness R(max) < 0.10 microm (s-Ti6Al4V) and a group with a rough surface with an R(max) < 0.25 microm (r-Ti6Al4V) were used. Titanium samples were immersed in SBF concentrated by five (SBF x 5) from 10 min to 5 h and examined by AFM and ESEM. Scattered Ca-P deposits of approximately 15 nm in diameter appeared after only 10 min of immersion in SBF x 5. These Ca-P deposits grew up to 60-100 nm after 4 h on both s- and r-Ti6Al4V substrates. With increasing immersion time, the packing of Ca-P deposits with size of tens of nanometers in diameter formed larger globules and then a continuous Ca-P film on titanium substrates. A direct contact between the Ca-P coating and the Ti6Al4V surface was observed. The Ca-P coating was composed of nanosized deposits and of an interfacial glassy matrix. This interfacial glassy matrix might ensure the adhesion between the Ca-P coating and the Ti6Al4V substrate. In the case of s-Ti6Al4V substrate, failures within this interfacial glassy matrix were observed overtime. Part of the glassy matrix remained on s-Ti6Al4V while part detached with the Ca-P film. The Ca-P coating detached from the smooth substrate, whereas the Ca-P film extended onto the whole rough titanium surface over time. In the case of r-Ti6Al4V, the Ca-P coating covered evenly the substrate after immersion in SBF x 5 for 5 h. The present study suggested that the heterogeneous nucleation of Ca-P on titanium was immediate and did not depend on the Ti6Al4V surface topography. The further growth and mechanical attachment of the final Ca-P coating strongly depended on the surface, for which a rough topography was beneficial.

Visualizing detergent resistant domains in model membranes with atomic force microscopy.

Evidence is accumulating that in cell membranes microdomains exist, also referred to as rafts or detergent resistant membranes. In this study, atomic force microscopy is used to study supported lipid bilayers, consisting of a fluid phosphatidylcholine, sphingomyelin and cholesterol. Domains were visualized of which the morphology and size depended on the cholesterol concentration. The presence of cholesterol was found to induce bilayer coupling. At 30 mol% cholesterol, a change in percolation phase was observed, and at 50 mol%, when both fluid lipids and solid lipids are saturated with cholesterol, phase separation was still observed. In addition, we were able to directly visualize the resistance of domains against non-ionic detergent.

Biomolecular interactions measured by atomic force microscopy.

Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites. In this review we discuss the important contributions that have led to the development of this field. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible measurements by AFM. This increased reproducibility will pave the way for a better understanding of molecular interactions in cell biology.

Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers.

We used atomic force microscopy (AFM) to study the lateral organization of transmembrane TmAW(2)(LA)(n)W(2)Etn peptides (WALP peptides) incorporated in phospholipid bilayers. These well-studied model peptides consist of a hydrophobic alanine-leucine stretch of variable length, flanked on each side by two tryptophans. They were incorporated in saturated phosphatidylcholine (PC) vesicles, which were deposited on a solid substrate via the vesicle fusion method, yielding hydrated gel-state supported bilayers. At low concentrations (1 mol %) WALP peptides induced primarily line-type depressions in the bilayer. In addition, striated lateral domains were observed, which increased in amount and size (from 25 nm up to 10 microm) upon increasing peptide concentration. At high peptide concentration (10 mol %), the bilayer consisted mainly of striated domains. The striated domains consist of line-type depressions and elevations with a repeat distance of 8 nm, which form an extremely ordered, predominantly hexagonal pattern. Overall, this pattern was independent of the length of the peptides (19-27 amino acids) and the length of the lipid acyl chains (16-18 carbon atoms). The striated domains could be pushed down reversibly by the AFM tip and are thermodynamically stable. This is the first direct visualization of alpha-helical transmembrane peptide-lipid domains in a bilayer. We propose that these striated domains consist of arrays of WALP peptides and fluidlike PC molecules, which appear as low lines. The presence of the peptides perturbs the bilayer organization, resulting in a decrease in the tilt of the lipids between the peptide arrays. These lipids therefore appear as high lines.

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion.

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy.

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

Mode of insertion of the signal sequence of a bacterial precursor protein into phospholipid bilayers as revealed by cysteine-based site-directed spectroscopy.

The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.

Mitochondrial presequence inserts differently into membranes containing cardiolipin and phosphatidylglycerol.

The interaction of the 25-residue presequence of yeast cytochrome oxidase subunit IV with lipid bilayers composed of phosphatidylglycerol, cardiolipin, or their (1:4) mixtures with phosphatidylcholine has been studied by spin-label ESR spectroscopy. Binding of the presequence progressively broadens the gel-to-fluid phase transition of dimyristoylphosphatidylglycerol bilayers, leading to abolition of the transition at a peptide/lipid ratio of > or = 1:5 mol/mol. The mobility of phosphatidylglycerol spin-labeled at the 5-position of the sn-2 chain is decreased in both gel and fluid phases on binding the presequence, with a progressively increasing ESR spectral anisotropy in the fluid phase. The ESR spectra of phosphatidylglycerol spin-labeled at the 14-position of the sn-2 chain contain a second motionally restricted component, in addition to the fluid bilayer spectral component, that arises from direct interaction of the bound presequence with the lipid chains. The proportion of this motionally restricted component is greater for dioleoylphosphatidylglycerol bilayers (corresponding to 2-3 lipids per peptide) than for cardiolipin bilayers (1-2 lipids/peptide), and this component is present also in the mixed bilayers containing 80% phosphatidylcholine. The ESR spectra of the presequence spin-labeled with a maleimide derivative at cysteine-19 evidence high mobility in solution and a very strong reduction in mobility on binding to bilayers containing negatively charged lipids. At low peptide to lipid ratios, the ESR spectra of the spin-labeled presequence sense the phase transition of dimyristoylphosphatidylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

SecA restricts, in a nucleotide-dependent manner, acyl chain mobility up to the center of a phospholipid bilayer.

The effects of SecA-lipid interactions on lipid mobility were studied by electron spin resonance (ESR) spectroscopy in bilayer systems containing phospholipids spin-labeled at different positions along the acyl chain. The SecA protein, which functions in protein translocation at the cytosolic side of the E. coli inner membrane, was found to decrease the mobility of the lipids upon its interaction with the membrane. The restriction of lipid motion, at all chain positions measured, reflects the ability of SecA to penetrate the membrane. At a 49:1 lipid/protein molar ratio, a second, motionally more restricted component is observed in ESR spectra of phospholipids spin-labeled close to the methyl ends of the chains (12th and 14th positions). Furthermore, SecA was found to eliminate the order-to-disorder phase transition of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol bilayers. A remarkably strong reduction in the ability of SecA to penetrate the membrane was found when the nucleotides ATP and ADP+P(i) were present. The presence of the non-hydrolyzable analogue AMP-PNP had no effect. These results clearly demonstrate that SecA perturbs, in a nucleotide dependent manner, lipid mobility upon insertion into the bilayer. The implications of these findings for translocation of precursor proteins across the E. coli inner membrane are discussed.

Membrane location of spin-labeled apocytochrome c and cytochrome c determined by paramagnetic relaxation agents.

The mitochondrial precursor protein horse heart apocytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region, and the mature protein yeast cytochrome c was similarly labeled on the single free cysteine residue at position 102 at the C-terminal. The proteins were bound to negatively charged phospholipid bilayers, and the accessibility of the spin-labeled cysteine residues to lipid-soluble molecular oxygen and to the lipid-impermeant chromium oxalate anion was determined from the saturation properties of the ESR spectra. Binding of the protein was found to have a considerable effect on the local oxygen concentrations within the lipid bilayer. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled either in the headgroup or at positions in the sn-2 acyl chain, in the presence of unlabeled protein, identify the position of the spin-labeled cysteine residues in the phospholipid bilayer. The spin label on apocytochrome c bound to phosphatidylglycerol bilayers lies between the 5- and 14-C positions of the lipid acyl chain. Admixture of > or = 75 mol % phosphatidylcholine induces an additional surface-associated apocytochrome c population. The spin label on native and heat-denatured cytochrome c is located at the membrane surface. These different extents of membrane penetration correlate also with the reduction in local oxygen concentration experienced by spin-labeled phospholipids on binding of apo- and holocytochrome c. The possible biological implications of the data are discussed.

Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.

Apocytochrome c derived from horse heart cytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region of the primary sequence, and cytochrome c from yeast was spin-labeled on the single cysteine residue at sequence position 102 in the C-terminal region. The spin-labeled apocytochrome c and cytochrome c were bound to fluid bilayers composed of different negatively charged phospholipids that also contained phospholipid probes that were spin-labeled either in the headgroup or at different positions in the sn-2 acyl chain. The location of the spin-labeled cysteine residues on the lipid-bound proteins was determined relative to the spin-label positions in the different spin-labeled phospholipids by the influence of spin-spin interactions on the microwave saturation properties of the spin-label electron spin resonance spectra. The enhanced spin relaxation observed in the doubly labeled systems arises from Heisenberg spin exchange, which is determined by the accessibility of the spin-label group on the protein to that on the lipid. It is found that the labeled cysteine groups in horse heart apocytochrome c are located closest to the 14-C atom of the lipid acyl chain when the protein is bound to dimyristoyl- or dioleoyl-phosphatidylglycerol, and to that of the 5-C atom when the protein is bound to a dimyristoylphosphatidylglycerol/dimyristoylphosphatidylcholine (15:85 mol/mol mixture. On binding to dioleoylphosphatidylglycerol, the labeled cysteine residue in yeast cytochrome c is located closest to the phospholipid headgroups but possibly between the polar group region and the 5-C atom of the acyl chains. These data determine the extent to which the different regions of the proteins are able to penetrate negatively charged phospholipid bilayers.

Interaction of spin-labeled apocytochrome c and spin-labeled cytochrome c with negatively charged lipids studied by electron spin resonance.

Apocytochrome c has been spin-labeled with a nitroxide derivative of maleimide on a cysteine residue at either position 14 or position 17 in the N-terminus. Yeast cytochrome c was spin-labeled with the same maleimide derivative on its single free cysteine residue at position 102 in the C-terminus. The ESR spectra of spin-labeled apocytochrome c have been characterized in different environments with respect both to the conformation of the protein and to its association with lipid. In buffer, the spectrum of spin-labeled apocytochrome c indicates high mobility, characteristic of the unfolded structure of the apoprotein, and that of spin-labeled cytochrome c is only slightly less mobile, suggesting that the site labeled is situated at the surface of the folded holoprotein. Upon binding the spin-labeled protein to negatively charged lipid membranes composed of dioleoylphosphatidylglycerol (DOPG), the ESR spectra of apocytochrome c evidence a large reduction in the mobility of the spin-label group, as also do those of yeast cytochrome c. In the case of apocytochrome c, this immobilization most likely arises from both an increase in secondary structure and a partial penetration of the protein into the lipid bilayer, in addition to the electrostatic interaction with the lipid headgroups, whereas for cytochrome c the immobilization observed arises primarily from an intimate association with the membrane surface. When the spin-labeled holocytochrome c is denatured by heating and is bound to DOPG bilayer membranes, a rather mobile ESR spectrum is observed, which demonstrates that the spin-label is located at the surface of the membrane in this case. The ESR spectra of spin-labeled apocytochrome c bound to mixed bilayers of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylcholine (DMPC) consist of both an immobile and a mobile component. The proportion of the mobile component is increased by increasing the mole fraction of the zwitterionic DMPC in the mixed bilayers. The mobile component represents a localization of apocytochrome c at the membrane surface, whereas the immobile component most probably represents the penetration of the precursor protein into the membrane interior. The immobile component assigned to membrane penetration of the precursor protein is still present at negatively charged lipid contents comparable to those in the native mitochondrial system. The results are discussed in relation to the conformation of apocytochrome c, its interaction with lipid, and the import of the apoprotein into mitochondria.

Accessibility of spin-labeled phospholipids in anionic and zwitterionic bilayer membranes to paramagnetic relaxation agents. Continuous wave power saturation EPR studies.

The location of phospholipids, spin-labeled in the headgroup or at various positions of the sn-2 chain, incorporated in bilayer membranes of dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol or dioleoylphosphatidylglycerol, has been calibrated in terms of their accessibility to paramagnetic relaxation agents. A power saturation approach has been used to determine the spin-label relaxation times, which in turn is influenced by spin-spin interactions with the different paramagnetic species. The effect of different paramagnetic relaxation agents on the power saturation behaviour of the spin-labeled lipids has been used to determine the relaxation enhancement which is quantified in terms of an accessibility parameter. Molecular oxygen, which dissolves preferentially in the lipid phase and the water-soluble, membrane-impermeant chromium oxalate anion are shown to report reliably on the accessibility of spin-labels located in one of the two phases. On the other hand, an uncharged, polar nickel-iminodiacetic acid complex is shown to enhance relaxation of spin-labels in both phases. These calibrations are essential for the study of the interaction of basic proteins with anionic lipid membranes.

Interaction of apocytochrome c and derived polypeptide fragments with sodium dodecyl sulfate micelles monitored by photochemically induced dynamic nuclear polarization 1H NMR and fluorescence spectroscopy.

The topology of apocytochrome c, the heme-free precursor of the mitochondrial protein cytochrome c, was investigated in a lipid-associated form. For this purpose photochemically induced dynamic nuclear polarization 1H nuclear magnetic resonance (CIDNP 1H NMR) spectroscopy and quenching of tryptophan and tyrosine fluorescence by acrylamide were applied to an apocytochrome c-sodium dodecyl sulfate (SDS) micellar system. A pH titration of the chemical shifts of the histidine C2 proton resonances of apocytochrome c, using conventional 1H NMR, yielded pK(a)'s of 5.9 +/- 0.1 and 6.2 +/- 0.1, which were assigned to histidine-18 and -33 and histidine-26, respectively. In the presence of SDS micelles an average pK(a) of 8.1 +/- 0.1 was obtained for all histidine C2 protons. Photo-CIDNP enhancements of the histidine, tryptophan, and tyrosine residues, contained in the intact apocytochrome c and in chemically and enzymatically prepared fragments of the precursor, were reduced in the presence of SDS micelles. Similarly, the quenching of the tryptophan fluorescence of the polypeptides by acrylamide was diminished in the presence of SDS. These results indicate the aromatic residues studied are localized in the interface of the SDS micelle.