Seth A. Darst

Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules

Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.

Three-dimensional structure of yeast RNA polymerase II at 16 Å resolution

The structure of yeast RNA polymerase II has been determined by three-dimensional reconstruction from electron micrographs of two-dimensional crystals at approximately 16 A resolution. The most prominent feature of the structure is an arm of protein density surrounding a channel about 25 A in diameter, similar to that found previously for E. coli RNA polymerase. The 25 A-diameter channel bifurcates on one face of the protein, connecting with a 25 A-wide groove and with a channel about half as wide. The 25 A channel and groove, and the narrow channel, may bind double- and single-stranded nucleic acids, respectively. A finger of protein density projecting from the molecule adjacent to the arm-like feature may represent the C-terminal domain of the largest subunit. These results provide a structural basis for analyses of the transcription process and its regulation.

Two-dimensional crystals of proteins on lipid layers

Proteins bound to lipid layers form single-layer (two-dimensional) crystals amenable to structure determination by electron microscopy and image processing. Recent studies have extended the range of the lipid-layer crystallization approach. In some cases, a simple electrostatic interaction between a protein and a charged lipid will suffice to direct binding, obviating the requirement for a specific lipid-binding ligand for two-dimensional crystal growth. Alternatively, streptavidin can be used to couple a biotinylated protein to a biotinylated lipid layer. Streptavidin itself forms large two-dimensional crystals that diffract to 2.8 A resolution, showing that ordering on lipid layers can be comparable to that in three dimensions.

Adsorption of the protein antigen myoglobin affects the binding of conformation-specific monoclonal antibodies

Five monoclonal antibodies against sperm whale myoglobin have been used to investigate the physical state of the antigen adsorbed onto a polydimethylsiloxane surface. The binding of each antibody is sensitive to the antigens conformation in solution while the locations of the antigenic sites on the myoglobin molecule for three of the antibodies have been determined (Berzofsky, J.A., G.K. Buckenmeyer, G. Hicks, F.R.N. Gurd, R.J. Feldmann, and J. Minna. 1982. J. Biol. Chem. 257:3189-3198). The binding of the fluorescein isothiocyanate-labeled IgG and Fab antibodies to previously adsorbed myoglobin has been observed using total internal reflection fluorescence. Three of the antibodies bind specifically to surface-adsorbed myoglobin with affinities at least 50% relative to myoglobin in solution whereas two of the antibodies show affinities for the surface-adsorbed myoglobin diminished by at least two orders of magnitude relative to myoglobin in solution. The specific loss of certain antigenic determinants on the adsorbed myoglobin, coupled with the retention of others, indicates a nonrandom adsorption of the myoglobin molecules.

Two-dimensional crystals of Escherichia coli RNA polymerase holoenzyme on positively charged lipid layers

Escherichia coli RNA polymerase holoenzyme forms two-dimensional crystals when adsorbed to positively charged lipid layers at the air/water interface. Adsorption of the protein is driven by electrostatic interactions between the positively charged lipid surface and the polymerase molecule, which has a net negative charge. Crystallization is dependent on the adsorption and concentration of RNA polymerase on fluid lipid surfaces. Image analysis of electron micrographs of crystals in negative stain, which diffract to 30 A resolution, shows irregularly shaped protein densities about 100 x 160 A, consistent with the dimensions of single polymerase molecules.

Bovine serum albumin adsorption and desorption rates on solid surfaces with varying surface properties

Abstract Total internal reflection fluorescence is used to examine the initial adsorption, desorption, and exchange kinetics of the protein bovine serum albumin (BSA) on six polymer surfaces with widely varying surface properties and functionalities. The six surfaces studied are polydimethylsiloxane (PDMS), polydiphenylsiloxane (PDϕS), polycyanopropylmethylsiloxane (PCPMS), polymethyl methacrylate (PMMA), polystyrene sulfonate (PSS), and polyethylene oxide (PEO). The results show that the initial adsorption of BSA on the surfaces PDMS, PDϕS, and PCPMS is diffusion limited up to wall shear rates of 4000 s −1 . The initial adsorption of BSA on PSS is diffusion limited at shear rates below about 70 s −1 but becomes kinetically controlled at higher shear rates. BSA adsorption on PMMA is kinetically controlled, and the adsorption of BSA on PEO is too slow to be detected under the conditions used. Studies on the kinetically limited BSA adsorption onto PMMA show that the adsorption process can be described by a kinetic rate expression that is first order in protein concentration. The initial desorption of BSA adsorbed onto each surface (except PEO) is shown to be kinetically limited. The rate-limiting step for the exchange of BSA adsorbed onto PDMS is shown to be the desorption of adsorbed BSA.

Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy

Electron crystallographic analysis of two-dimensional crystals grown on lipid layers at the air/water interface has been limited by loss or damage during transfer of the crystals to an electron microscope support grid. Two methods of transfer are described which are applicable on a small scale (10 microliters of protein solution) and which give greatly improved results for streptavidin crystals on biotinylated lipid layers. In the first method, a hydrophobic grid surface was produced by coating a carbon support film with a thin layer of SiO2, followed by alkylation with dimethyloctadecylchlorosilane. The transfer efficiency of protein crystals approached 50% coverage of the alkylated grid surface. The degree of order of crystals transferred to the alkylated grid surface and preserved in negative stain was significantly improved over that of crystals transferred directly to a carbon support film. In the second method, crystals at the air/water interface were transferred to a holey carbon support film. The efficiency of transfer across the holes was virtually 100% as nearly every hole was completely covered with crystals. After preservation of the crystals in 1% glucose and cooling to liquid nitrogen temperature, electron diffraction was obtained that extended to 1/2.8 A-1 resolution. This demonstrates that two-dimensional crystals grown on lipid layers at the air/water interface can be sufficiently well-ordered, even after transfer to a support grid, to yield high-resolution structural information.

Two-dimensional and epitaxial crystallization of a mutant form of yeast RNA polymerase II

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.

Structural study of the yeast RNA polymerase A: Electron microscopy of lipid-bound molecules and two-dimensional crystals

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.

Myoglobin adsorption onto crosslinked polydimethylsiloxane

Abstract The adsorption of sperm whale myoglobin (Mb) on crosslinked polydimethylsiloxane (silicone rubber) from flowing solutions is examined using total internal reflection fluorescence (TIRF). By comparing the experimentally observed relationship between the adsorption rate and the wall shear rate with the predictions of a convection/diffusion model, it is shown that the initial adsorption is diffusion-controlled over the observed range of wall shear rates (49 to 410 s −1 ). As time proceeds, the fluorescence intensity rises rapidly and begins to plateau. This is followed by another rapid rise. This “shoulder” in the fluorescence intensity-time relationship is not an artifact emanating from extraneous bulk solution fluorescence. The fluorescence intensity at which the shoulder occurs is independent of the Mb solution concentration. Over long periods of time (several hours), a slow increase in the fluorescence intensity is observed which is not due to the adsorption of additional protein and is also not caused by long-term exposure of the surface to the laser radiation. Experiments investigating the adsorption of FITC-labeled immuno-γ-globulin in solution to adsorbed layers of unlabeled Mb indicate that the time course of the long-term fluorescence quantum yield change corresponds to the spreading of surface adsorbed Mb to fill in vacant regions on the PDMS surface. The filling of vacant regions and the long-term quantum yield increase are considered to be manifestations of conformation changes of surface adsorbed Mb.