Steven G. Boxer

Phase Separation of Lipid Membranes Analyzed with High-Resolution Secondary Ion Mass Spectrometry

Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of ∼100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.

Advances in imaging secondary ion mass spectrometry for biological samples

Imaging mass spectrometry combines the power of mass spectrometry to identify complex molecules based on mass with sample imaging. Recent advances in secondary ion mass spectrometry have improved sensitivity and spatial resolution, so that these methods have the potential to bridge between high-resolution structures obtained by X-ray crystallography and cyro-electron microscopy and ultrastructure visualized by conventional light microscopy. Following background information on the method and instrumentation, we address the key issue of sample preparation. Because mass spectrometry is performed in high vacuum, it is essential to preserve the lateral organization of the sample while removing bulk water, and this has been a major barrier for applications to biological systems. Recent applications of imaging mass spectrometry to cell biology, microbial communities, and biosynthetic pathways are summarized briefly, and studies of biological membrane organization are described in greater depth.

Molecular transport and organization in supported lipid membranes

The mechanism by which vesicles spontaneously form supported lipid bilayer membranes on glass surfaces is becoming better understood and this knowledge is the basis of a technology of patterning membrane arrays and controlling composition. Controlled interactions between supported membranes and cells, particularly from the immune system, provide direct insight into cell-cell surface interactions.

Early Steps of Supported Bilayer Formation Probed by Single Vesicle Fluorescence Assays

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.

Dielectric Asymmetry in the Photosynthetic Reaction Center

Although the three-dimensional structure of the bacterial photosynthetic reaction center (RC) reveals a high level of structural symmetry, with two nearly equivalent potential electron transfer pathways, the RC is functionally asymmetric: Electron transfer occurs along only one of the two possible pathways. In order to determine the origins of this symmetry breaking, the internal electric field present in the RC when charge is separated onto structurally characterized sites was probed by using absorption band shifts of the chromophores within the RC. The sensitivity of each probe chromophore to an electric field was calibrated by measuring the Stark effect spectrum, the change in absorption due to an externally applied electric field. A quantitative comparison of the observed absorption band shifts and those predicted from vacuum electrostatics gives information on the effective dielectric constant of the protein complex. These results reveal a significant asymmetry in the effective dielectric strength of the protein complex along the two potential electron transfer pathways, with a substantially higher dielectric strength along the functional pathway. This dielectric asymmetry could be a dominant factor in determining the functional asymmetry of electron transfer in the RC.

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Synthetic lipid–oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5′-coupled conjugate and the other the 3′-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane–membrane spacing on fusion, a series of conjugates was constructed by adding 2–24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.

Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI’s rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme’s catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems. Vibrational spectroscopy pinpoints a surprisingly large local electric field where an enzyme binds its substrate. [Also see Perspective by Hildebrandt] Stark influence on reaction rates Enzymes accelerate chemical processes by coaxing molecules into just the right reactive states. Fried et al. now elucidate the way the enzyme ketosteroid isomerase pushes its substrate toward product through exertion of a local electric field (see the Perspective by Hildebrandt). First the authors calibrated the shifts in molecular vibrational frequencies, known as Stark shifts, that fields of varying strength impose on a substrate analog; then they measured the vibrational spectrum of that compound in the enzymes active site. The experiment uncovered an unusually strong field that the local enzyme structure directed to the precise spot where the substrate would react. Science, this issue p. 1510; see also p. 1456

Cell adhesion to protein-micropatterned-supported lipid bilayer membranes

A new method for constructing controlled interfaces between cells and synthetic supported lipid bilayer membranes is reported. Microcontact printing is used to define squares and grid lines of fibronectin onto glass, which subsequently direct the self-assembly of fluid lipid bilayers onto the complementary, uncoated regions of the surface. Features of fibronectin as small as 5 microm effectively control the lateral organization of the lipid bilayers. These fibronectin barriers also facilitate the adhesion of endothelial cells, which exhibit minimal adhesion to fluid supported lipid bilayers alone. Cells selectively adhere to the features of fibronectin, spanning over and exposing the cells to the intervening regions of supported lipid bilayer. Cell spreading is correlated with both the geometry and dimensions of the fibronectin barriers. Importantly, lipids underlying adherent cells are laterally mobile, suggesting that, in contrast to the regions of fibronectin, cells were not in direct contact with the supported membrane. Protein micropatterning thus provides a valuable tool for controlling supported membranes and for juxtaposing anchorage-dependent cells with lipid bilayers. These systems should be generally useful for studying specific interactions between cells and biomolecules incorporated into supported membranes, and as an approach for integrating living cells with synthetic, laterally complex surfaces.

Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen-Bonding Effects

Infrared (IR) band shifts of isolated vibrational transitions can serve as quantitative and directional probes of local electrostatic fields, due to the vibrational Stark effect. However, departures from the Stark model can arise when the probe participates in specific, chemical interactions, such as direct hydrogen bonding. We present a method to identify and correct for these departures based on comparison of (13)C NMR chemical shifts and IR frequencies each calibrated in turn by a solvatochromic model. We demonstrate how the tandem use of these experimental observables can be applied to a thiocyanate-modified protein, ketosteroid isomerase, and show, by comparison to structural models, that changes in electrostatic field can be measured within the complex protein environment even in the background of direct hydrogen bonding to the probe.

Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag

A very rapid method for isolating bacterial photosynthetic reaction centers (RCs) of high purity and yield is described. A poly-histidine tag is engineered at the C-terminus of the RC M-subunit, allowing recovery of pure RCs in less than 4 h with a commercially available Ni2+ resin. The phenotype of the protein including absorption spectra and electron transfer kinetics are virtually identical to wild-type RCs. This method should facilitate studies of RCs by dramatically decreasing sample preparation time and eliminating the use of expensive equipment.