Biomedical subjects

W R Schief

Publications and source records attributed to W R Schief.

7 recordsLinked to original sources

PubMed2001

Discrepancy between phase behavior of lung surfactant phospholipids and the classical model of surfactant function.

The studies reported here used fluorescence microscopy and Brewster angle microscopy to test the classical model of how pulmonary surfactant forms films that are metastable at high surface pressures in the lungs. The model predicts that the functional film is liquid-condensed (LC) and greatly enriched in dipalmitoyl phosphatidylcholine (DPPC). Both microscopic methods show that, in monolayers containing the complete set of phospholipids from calf surfactant, an expanded phase persists in coexistence with condensed domains at surface pressures approaching 70 mN/m. Constituents collapsed from the interface above 45 mN/m, but the relative area of the two phases changed little, and the LC phase never occupied more than 30% of the interface. Calculations based on these findings and on isotherms obtained on the continuous interface of a captive bubble estimated that collapse of other constituents increased the mol fraction of DPPC to no higher than 0.37. We conclude that monolayers containing the complete set of phospholipids achieve high surface pressures without forming a homogeneous LC film and with a mixed composition that falls far short of the nearly pure DPPC predicted previously. These findings contradict the classical model.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

Conformational changes during kinesin motility.

Nucleotide-dependent movements of the head and neck of kinesin have been visualized by cryoelectron microscopy and have been inferred from single-molecule studies. Key predictions of the hand-over-hand model for dimeric kinesin have been confirmed, and a novel processivity mechanism for the one-headed, kinesin-related motor KIF1A has been discovered.

Animals↗

PubMed1999

Two-dimensional crystallization of streptavidin: in pursuit of the molecular origins of structure, morphology, and thermodynamics.

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.

Crystallization↗

PubMed1999

Phase separation in monolayers of pulmonary surfactant phospholipids at the air-water interface: composition and structure.

The phase behavior of monolayers containing the complete set of purified phospholipids (PPL) obtained from calf surfactant was investigated as a model for understanding the phase transitions that precede compression of pulmonary surfactant to high surface pressure. During compression, both fluorescence microscopy and Brewster angle microscopy (BAM) distinguished domains that separated from the surrounding film. Quantitative analysis of BAM grayscales indicated optical thicknesses for the PPL domains that were similar to the liquid condensed phase for dipalmitoyl phosphatidylcholine (DPPC), the most abundant component of pulmonary surfactant, and higher and less variable with surface pressure than for the surrounding film. BAM also showed the optical anisotropy that indicates long-range orientational order of tilted lipid chains for the domains, but not for the surrounding film. Fluorescence microscopy shows that addition of DPPC to the PPL increased the area of the domains. At fixed surface pressures from 20-40 mN/m, the total area of each phase grew in proportion with the mol fraction of DPPC. This constant variation allowed analysis of the DPPC mol fraction in each phase, construction of a simple phase diagram, and calculation of the molecular area for each phase. Our results indicate that the phase surrounding the domains is more expanded and compressible, and contains reduced amounts of DPPC in addition to the other phospholipids. The domains contain a mol fraction for DPPC of at least 96%.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Electron crystallographic analysis of two-dimensional streptavidin crystals coordinated to metal-chelated lipid monolayers.

Coordination of individual histidine residues located on a protein surface to metal-chelated lipid monolayers is a potentially general method for crystallizing proteins in two dimensions. It was shown recently by Brewster angle microscopy (BAM) that the model protein streptavidin binds via its surface histidines to Cu-DOIDA lipid monolayers, and aggregates into regularly shaped domains that have the appearance of crystals. We have used electron microscopy to confirm that the domains are indeed crystalline with lattice parameters similar to those of the same protein crystallized beneath biotinylated lipid monolayers. Although BAM demonstrates that the two-dimensional protein crystals grown via metal chelation are distinct from the biotin-bound crystals in both microscopic shape and thermodynamic behavior, the two crystal types show similar density projections and the same plane group symmetry.

Adsorption↗

PubMed1996

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Bacterial Proteins↗

PubMed1996

Lateral phase separation in interfacial films of pulmonary surfactant.

To determine if lateral phase separation occurs in films of pulmonary surfactant, we used epifluorescence microscopy and Brewster angle microscopy (BAM) to study spread films of calf lung surfactant extract (CLSE). Both microscopic methods demonstrated that compression produced domains of liquid-condensed lipids surrounded by a liquid-expanded film. The temperature dependence of the pressure at which domains first emerged for CLSE paralleled the behavior of its most prevalent component, dipalmitoyl phosphatidylcholine (DPPC), although the domains appeared at pressures 8-10 mN/m higher than for DPPC over the range of 20-37 degrees C. The total area occupied by the domains at room temperature increased to a maximum value at 35 mN/m during compression. The area of domains reached 25 +/- 5% of the interface, which corresponds to the predicted area of DPPC in the monolayer. At pressures above 35 mN/m, however, both epifluorescence and BAM showed that the area of the domains decreased dramatically. These studies therefore demonstrate a pressure-dependent gap in the miscibility of surfactant constituents. The monolayers separate into two phases during compression but remain largely miscible at higher and lower surface pressures.

Animals↗