HPI Layer Research Articles

Stability of S-layer proteins for electrochemical nanofabrication

Crystalline cell surface layer proteins (S-layers) can be used in electrochemical fabrication to create nanoscale arrays of metals and oxides on surfaces so long as the proteins maintain their long-range order during processing. We have explored the stability of the HPI layer protein (the S-layer protein from the microorganism Deinococcus radiodurans) adsorbed onto platinum surfaces after immersion in sulfuric acid or sodium hydroxide electrolytes ranging in pH from 0 to 14 over time periods ranging from 1 to 1000 s. Topographic data obtained by atomic force microscopy (AFM) was used to characterize the protein stability, judged by its retention of long-range order after immersion. The compiled data revealed that, under these solution conditions and in this environment, the HPI layer protein has a dose-dependent structural stability “envelope” in the acidic range from 1 < pH < 4. The protein retains its long-range order up to 1000 s from pH 4 to 11, and has a sharp stability edge between pH 12 and 13. Interestingly, the more stringent requirement of stability ( i.e., retention of long-range order) defined in the context of electrochemical fabrication for this protein narrowed the window of stability in pH and time when compared to previous stability studies reported for this protein.

In Vivo Atomic Force Microscopy of Surface Proteins on Deinococcus radiodurans

We present in vivo images of the outer cell wall of the bacteria Deinococcus radiodurans using noncontact atomic force microscopy (AFM). High-resolution imaging shows a hexagonal-packed lattice. This lattice is consistent with previous observations of a hexagonally packed intermediate layer from extracted cell wall material. The ordered hexagonal structures were primarily observed at the interfaces between adjoining bacteria, while rougher, orange-peel-like structures were observed in other regions. It is possible that the bacteria were in a dormant state during imaging as the desiccation tolerance of D. radiodurans is well documented. The only previous observations of the HPI layer of D. radiodurans have been performed on chemically extracted S-layers applied to flat substrates. Our report is the first in vivo observation of these structures in D. radiodurans without invasive sample preparation. Additionally, this is the first known report of in vivo observation of bacterial S-layers by AFM.

Hydration Scanning Tunneling Microscopy of DNA and a Bacterial Surface Protein

Hydration scanning tunneling microscopy is based on the electrical conductivity of molecularly thin water layers which adsorb to the sample surfaces in a humid atmosphere. It allows reliable imaging of biological specimens and even insulators, provided they are hydrophilic. Here, we present results obtained with linearized plasmid DNA on mica and a bacterial surface protein layer (the HPI layer). A width of 3 nm was measured for the DNA molecules and a quasi-periodic structure along the molecules with a repeat distance of about 5 nm was observed. We show that—depending on the tunneling voltage—there are two different imaging modes for the DNA samples: at higher voltages, real tunneling or field emission is responsible for the charge transfer between tip and sample. In contrast, at lower voltages we found indications of a water meniscus between tip and surface. The HPI layer, however, seems to be imaged at most voltages without a water meniscus.

Atomic force microscopy of a hydrated bacterial surface protein

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.

Comparative study of a regular protein layer by scanning tunneling microscopy and transmission electron microscopy

Scanning tunneling microscopy (STM) imaging has been performed on the HPI layer (hexagonally packed intermediate layer), freeze-dried and coated with Pt–Ir–C. The HPI layer is a highly corrugated regular protein monolayer, found outermost in the cell wall of the bacterium Deinococcus radiodurans. The average structure of each of the two surface aspects of HPI layer has been determined from STM data using correlation averaging. The signal-to-noise ratio of the raw data obtained from STM of the HPI layer is in the range of 1.5 to 2, values much higher than those usually encountered in data obtained by transmission electron microscopy (TEM). In order to discuss reliability and accuracy of STM imaging, the reliefs obtained by STM are compared to reconstructed reliefs derived from TEM micrographs of freeze-dried and metal-coated specimens.

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.

Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans

The low-resolution structure of the regular surface layer of Deinococcus radiodurans has been determined from negatively stained specimens by three-dimensional electron microscopy. The layer has P6 symmetry, a lattice constant of 18 nm and a thickness of 6.5 nm. Three-dimensional reconstruction was performed by a hybrid real space/Fourier space approach that incorporates partial compensation of lattice distortions. The model obtained is discussed in the light of independent information about the surface structure of this layer, derived from metal shadowing and surface relief reconstruction. While agreement is quite satisfactory for the apparently more rigid inner surface, the outer surface shows severe flattening effects. The structure of the HPI layer is compared with other bacterial surface layers using a classification scheme that is outlined in the Appendix.

Projected structure of the surface protein of deinococcus radiodurans determined to 8 Å resolution by cryomicroscopy

Cryomicroscopy at liquid nitrogen temperature has been used, in conjunction with correlation averaging, to determine the projection structure of the surface protein (HPI layer) of the Bacterium Deinococcus radiodurans to a resolution of 0.83 nm. The two-dimensional crystals were embedded in a thin layer of frozen-hydrated aurothioglucose, obtained by the controlled evaporation of an aurothioglucose solution and cryofixation in the syrup state. The relative merits of this preparation procedure are evaluated.

Three-dimensional reconstruction of a freeze-dried and metal-shadowed bacterial surface layer

Three-dimensional reconstructions were performed from tilt series of a rotary-shadowed regular bacterial surface layer. Two series, representing the two surfaces of the HPI layer, were processed. The metal film stands out very clearly from the surrounding matter (protein, carbon) in the reconstructions and can, in good approximation, be regarded as reproducing the surface geometry of the protein layer. Thus, by combining information from both surfaces, it is possible to reconstruct profiles of the protein structure. Shadowing is advantageous in that it provides excellent reproducibility in specimen contrast, and the metal film is stable in the electron beam. Moreover, unlike negative staining, information is obtained from surfaces that are not in contact with the specimen support, and the technique is also applicable to freeze-dried material.

High resolution shadowing with pure carbon

Two test specimens, isolated purple membrane sheets of Halobacterium halobium and HPI layer of Deinococcus radiodurans, were used to investigate the condensation behaviour of pure carbon evaporated onto freeze-dried biological material. Carbon was evaporated at 15°, 45° and 90° elevation angle with, or without, rotating the specimen. On the exoplasmatic surface of the purple membrane a relief structure was found, causing a condensation pattern which correlates with the position of the bacteriorhodopsin molecules in a similar way as the decoration sites shown by Studer et al. [J. Cell Biol. 90 (1981) 153] after tantalum/tungsten shadowing. The very detailed relief structure of the HPI layer is also well portrayed by shadowing with pure carbon; the results are comparable to heavy metal shadowings shown by Wildhaber et al. [Ultramicroscopy 16 (1985) 411]. C-shadowing is a method complementary to heavy metal shadowing: Carbon condenses more or less amorphously. Because the deposition of carbon is enhanced in holes and depressions due to partial reflection effects, the resulting images resemble in some respect those from negatively stained preparation. The contrast of C-shadowed specimens is sufficiently high; no low-dose techniques have to be used.

High resolution metal replication, quantified by image processing of periodic test specimens

With the aid of periodic test specimens we studied the information contained in metal replica films, used for contrasting biological material. The so-called HPI layer, a protein monolayer of the cell wall of the bacterium Deinococcus radiodurans and the purple membrane of Halobacterium halobium were examined by freeze-drying (“surfaces”). The sheet-like structures were directly prepared on carbon-coated grids. The paracrystalline regions of the plasmalemma of baker's yeast were studied by means of freeze-fracturing (“fracture faces”). The influence of film thickness, shadowing material (Pt/C,Ta/W),specimen temperature during shadowing and the differences between unidirectional and rotary shadowing were evaluated. For shadowing at very low specimen temperature (-260°C) an ultra-high vacuum ( p⩽10 -9 mbar) machine has been developed. The machine is equipped with a high vacuum specimen lock, allowing ultra-high vacuum experiments to be routinely performed.

New high resolution imaging methods as tools for the observation of small clusters

With high resolution imaging under conditions of short-range phase contrast noise it is possible to visualize small heavy-metal clusters even on a relatively thick biological structure. In the present study the surface of the HPI layer has been studied after rotary-shadowing it with a 5 Å thick Ta/W film. Clusters of 10 Å and less in size are observed within an arbitrarily chosen unit cell. The visualization of clusters allows one to quantify the resolution of shadowing films and the decoration effect of metal clusters.

The effects of air-drying and freeze-drying on the structure of a regular protein layer

The surface structure of the HPI layer of Deinococcus radiodurans is determined by metal shadowing. Particular attention is paid to the effects exerted on this structure by dehydration. Comparison of air-dried and freeze-dried preparations allows a phenomenological description of the dehydration artefacts. Analysis with correlation techniques reveals that apart from systematic effects (like a flattening of the structure) unprotected drying increases structural heterogeneity.

Decoration effects on the surface of a regular protein layer

The analysis of high resolution metal films, evaporated under decoration conditions, yields supplementary information on the biological specimen when compared with shadowing. The decorating metal clusters reveal the physico-chemical properties of the substrate structure depending on varying interactions of the film material on the surface. Image processing methods are used to quantify the cluster arrangement. An attempt is made to localize the preferred nucleation sites for evaporated platinum and silver on the protein surface of HPI layer.

Textural image processing on granular structures of metal shadowing replicas

Grain measurements are proposed in order to quantitatively specify the grain characteristics of metal shadowing replicas. The measures suggested were implemented by computer routines and both demonstrated with the help of computer-generated grain models and tested on several replicas of the HPI layer. An attempt is made to calculate the statistics due to the lateral mobility of the grains during the shadowing procedure.

The major cell envelope protein of Micrococcus radiodurans (R1). Structural and chemical characterization.

The so‐called HPI layer, representing the major cell envelope protein of the radiotolerant bacterium Micrococcus radiodurans, is a hexagonally patterned macromolecular monolayer; it is intimately associated with the outer membraneous layer (‘backing layer’) of the bacterium and covers its surface completely. By virtue of its quite unusual resistance to chemical perturbants it can easily be isolated, e.g. by detergent extraction of whole cells.A detailed description of the morphology of the HPI layer protein is given (resolution 1.8 nm), based on electron microscopy and image processing. Infrared spectroscopy shows that the secondary structure of the protein is stable under a wide range of environmental conditions; a considerable portion (30%) of the protein exists in the form of B structure.Direct mass measurements in a scanning transmission electron microscope reveal that the HPI layer protein has a molecular mass of approximately 655 kDa. Although masked by extensive proteolytic cleavage it appears that the morphologicaI protein units are composed of six identical protomers. All evidence accumulated so far supports our view that proteolysis is not a preparation artifact, but occurs already in vivo, without affecting the physical structure of the protein.

Mass mapping of a protein complex with the scanning transmission electron microscope.

A mass map of the hexagonally packed intermediate layer (HPI-layer), a regular protein monolayer from the cell envelope of Micrococcus radiodurans, has been obtained by scanning transmission electron microscopy. Samples were freeze-dried within the microscope, and low-dose images were recorded in the dark-field mode directly in digital form and processed by correlation averaging. The averaged projection of the unstained structure--i.e., the mass map--thus calculated shows a resolution to 3-nm period and reveals morphological features consistent with those obtained by negative staining. The mass of individual morphological domains was extracted by using variously the mass map itself or an average from a negatively stained HPI layer to define the domain boundaries. Protrusions as small as 1,300 daltons could be measured reproducibly within the unit cell of 655,000 daltons. The method developed opens an avenue to identify molecular species in situ and to correlate topographic information with biochemical data.

The structure of the cell envelope of Micrococcus radiodurans as revealed by metal shadowing and decoration

The cell envelope of Micrococcus radiodurans has been examined by Ta/W shadowing complemented by Ag decoration. Particular attention was paid to the structure determination of the HPI layer, a hexagonally patterned monolayer of protein, and its association with the outer membranous layer of the bacterium. By combining the information from the several structural aspects found, a model is proposed for the structure of the HPI-layer protein and its orientation in the cell envelope is established.

Topographic study of the cell surface of micrococcus radiodurans.

The paracrystalline outer membraneous layer (HPI layer) of Micrococcus radiodurans has been investigated by negative- and positive-staining electron microscopy and subsequent digital image processing. The subunit structure of the major HPI layer protein complex and the lipid-protein distribution in the plane of the membrane have been determined. The HPI layer was found to be highly asymmetric in a transmembrane direction, with the major protein complex only partly penetrating into a lipid-containing backing layer intimately associated with it.