Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy

Devaud, Genevieve; Furcinitti, Paul S.; Fleming, James C.; Lyon, Mary K.; Douglas, Kenneth T.

doi:10.1016/s0006-3495(92)81651-x

Cited by 30 publications

(19 citation statements)

References 14 publications

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“…have a type of cell envelope found only among Archaea, in which the sole cell wall component is a paracrystalline surface layer (S-layer). Electron-microscopic and scanning-probe studies show that these S-layers have regularly arrayed, well-defined pores and topologically distinct inner and outer faces (Deatherage et al 1983;Devaud et al 1992). Though composed of protein, S-layers of Sulfolobus spp.…”

Section: Introductionmentioning

confidence: 99%

“…Though composed of protein, S-layers of Sulfolobus spp. are stable in sodium dodecyl sulfate (SDS) or other detergents, which facilitates their isolation from whole cells by repeated detergent extraction and centrifugation (Weiss 1974;Michel et al 1980;Devaud et al 1992). The resulting baglike structures (sacculi) can be solubilized by adjusting the pH, and the constituent protein subunits can be analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) (Michel et al 1980;Grogan 1989).…”

Section: Introductionmentioning

confidence: 99%

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Organization and interactions of cell envelope proteins of the extreme thermoacidophileSulfolobus acidocaldarius

Grogan¹

1996

Can. J. Microbiol.

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To address basic questions concerning proteins of the Suljolobus acidocaldarius cell1 envelope, cell ghosts (empty cells consisting of cytoplasmic membrane complexed with the glycoprotein S-layer) were iso1;xted and analyzed by standard biochemical methods. The constituent proteins included the two dissimilar subunits also founcl in the baglike structures (sacculi) of S-layer (purified from whole cells by detergent extraction), plus approximately 30 additional protein species. When S-layer-dissociating conditions were applied to intact cell ghosts, the large sacculus subunit was released, whereas the small subunit remained bound to the cytoplasmic membrane. These results provided evidence that the small subunit may anchor the S-layer to the S. acidocaldarius cell membrane. The S. acidocaldarius S-layer was highly resistant to proteolysis in its native state but not when dissociated into subunits. Virtually all other membrane-associated proteins were readily digested by pronase treatment of cell ghosts. Pronase treatment of whole cells, in contrast, removed only a defined subset of the membrane-associated proteins, suggesting that this subset represents a few proteins nonnally exposed to the external medium. Several putative membrane glycoproteins were detected in cell ghosts that were not demonstrably associated with the S-layer.Resume : Pour rkpondre B certaines questions fondamentales sur les protkines de l'enveloppe cellulaire du Suljolobus acidocaldarius , nous avons is016 des ccfant6mes~ de cellules (cellules vides comprenant la membrane cytoplasmique complex6e avec la glycoprotkine de la couche S) et les avons analysks par les mkthodes biochiimiques usuelles. Les protkines constituantes comprennent deux sous-unit6s non-semblables aussi retrouvkes dans des structuires en forme de sac (saccules) de la couche S (purifike B partir de cellules entibres par extraction avec des dktergents), ainsi qu'environ 30 autres sortes de prot6ines. Lorsque les fant6mes cellulaires sont placCs dans des conditions permettant de dissocier la couche S, la grosse sous-unit6 du saccule est detachke, mais la petite sous-unit6 demeure like B la membrane cytoplasmique. Ces rksultats confirment que la petite sous-unit6 peut accrocher la couche S B la membrane cellulaire du S. izcidocaldarius. La couche S du S. acidocaldarius est formement rksistante B la protColyse B 1'Ctat nature1 mais pas lorsqu'elle est scind6e en sous-unit6s. Toutes les autres protkines membranaires sont rapidement digkrkes par un traitment 2 la pronase des fant6mes de cellules. Un m&me traitement sur des cellules entibres n'enlbve qu'une portion prkcise de l'assemblage des protkines associkes 2 la membrane suggCrant que cette portion ne reprksente que quelques protkines normalement exposkes au milieu exteme. Quelques glycoprotkines membranaires putatives ont Ctk dktect6es dans les fant6mes de cellules, mais leur association avec la couche S n'a pas 6t6 dkmontrke.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Organization and interactions of cell envelope proteins of the extreme thermoacidophileSulfolobus acidocaldarius

Grogan¹

1996

Can. J. Microbiol.

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“…The enzymes on plasma membranes were more active than those on vacuole membranes. In view of relevant theories, graphic models of biomembranes [11][12][13][14]16,19,[24][25][26][27][28] , the study of the chemical components of the plasma surface of winter wheat in this work (proteolytic enzyme treatment experiments) and our previous work on cytochemistry of cell membranes, an assumption was made that the protrusions on the plasmalemma surfaces were likely to be membrane proteins. To verify the speculation, proteinase digestion experiment was carried out.…”

Section: Topography Of Plasma Membrane Imaged By Afmmentioning

confidence: 99%

“…In recent years, AFM has been used to investigate cell surfaces, biomembranes, membrane proteins and glycolipids. Great progress has been made with the application of this efficient experimental tech--nique [11][12][13][14][15][16][17][18][19][24][25][26][27][28] . Karrasch used AFM to produce faithful high-resolution images of proteins on the surfaces of the hexaganally packed intermediate layer (HPI) of Deinococcus radiodurans with a lateral resolution of 1 nm and a vertical resolution of 0.1 nm [11] .…”

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confidence: 99%

Topography and functional information of plasma membrane

Sun

Chen

Song

et al. 2008

Sci. China Ser. C-Life Sci.

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By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8+/-0.09 nm in diameter and 1.9+/-0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40-60 nm, and a range of 1.8-5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12-40 nm for their diameter and 0.7-2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 microm(2). According to their size, we classified the invaginated pits into two types--larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead protoplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still detectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in diameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6-9.8 nm, thicker in regions covered with proteins.

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“…Protein images have also been obtained and reported, but there are many problems to imaging of proteins by the contact mode AFM (e.g., Butt et al 1990, Devaud et al 1992, Karrasch et al 1993, Ohnishi et al 1992, Wigren et al 1991, Yang et al 1994. Scratching of the sample surface with the AFM tip is the most serious problem.…”

Section: Introductionmentioning

confidence: 99%

The structure difference of proteins isolated on substrate with different techniques as studied by the atomic force microscope

et al. 1996

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Summary:The height and width of proteins deposited on mica substrates were measured from cross sections of their atomic force microscope (AFM) images. The tapping mode AFM gave very stable and reproducible images for high molecular weight proteins. The following results were obtained:(1) The thickness of mono-, bi-, and trilayered purple membranes was 5.3, 10.4, and 16.0 nm, respectively. The calibration curve of z range of AFM based on the above data was linear. The deviation of the calibration curve at the origin was < 0.6 nm. (2) The height of slow form α-2-macroglobulin (α2M) molecule changed depending on sampling methods. When the protein was freeze-dried on a mica substrate prewetted with water, α2M gave the highest value for its height. The fact that freeze-drying, especially after prewetting of the substrate, was effective to prevent flattening of the molecule suggested that sample deposition must be as gentle as possible to keep the original height of the molecules. (3) Furthermore, we compared differences of height and width between α2M and myosin filament. The result suggested that α2M had a disklike rather than a spherical form. Large proteins such as α2M are still difficult to crystallize for x-ray analysis, and we tested the AFM method for the study of minute height differences of such proteins.

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Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy

Cited by 30 publications

References 14 publications

Organization and interactions of cell envelope proteins of the extreme thermoacidophileSulfolobus acidocaldarius

Organization and interactions of cell envelope proteins of the extreme thermoacidophileSulfolobus acidocaldarius

Topography and functional information of plasma membrane

The structure difference of proteins isolated on substrate with different techniques as studied by the atomic force microscope

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