Gerrit Oostergetel

THE CRYSTALLINE DOMAINS IN POTATO STARCH GRANULES ARE ARRANGED IN A HELICAL FASHION

Abstract The structural basis for the physical properties of starches from different botanical sources is still poorly understood. Particularly at the level of the crystalline domains the present knowledge concerning the structure of starch is limited. This paper reports the semi-crystalline structure of potato starch granules by electron optical tomography and by cryo electron diffraction. It is concluded that the crystalline domains in the amylopectin form a continuous network of left-handed helices, which appears as a well-ordered skeleton for the starch granule. This type of super-helical lamellar structure has not been reported before, either in natural or in synthetic polymers.

Automation of specimen selection and data acquisition for protein electron crystallography

Abstract A system is presented for semi-automatic specimen selection and data acquisition for protein electron crystallography, based on a slow-scan CCD camera connected to a transmission electron microscope and control from an external computer. Areas of interest on the specimen are localised at low magnification and subsequently imaged on the CCD camera, using a dose which is small compared to the dose used in the exposure mode. The crystalline quality of the area is evaluated from the appearance of diffraction peaks in the calculated image Fourier transform. If the quality is considered good, images can then be recorded in different modes, both on film and using the CCD camera. Using this system a significant gain, both quantitatively and qualitatively, can be obtained in acquiring data for electron crystallography of beam-sensitive materials.

Recrystallization of amylopectin in concentrated starch gels

The relation between the recrystallization of amylopectin and the increase in stiffness of starch gels during storage was studied by various techniques. From transmission electron microscopy it was concluded that the size of the crystalline domains in retrograded 30% w/w potato starch gels was about 5 nm, much smaller than those present in native starch. The super-helical structure formed by the crystalline domains in native starch granules was not seen. It may thus be concluded that in retrograded starch gels the long range ordering is not regained during retrogradation. The relation between the degree of recrystallization, as determined with differential scanning calorimetry (DSC), and the increase in stiffness, as measured in compression, was found to be closely related. Two mechanisms will be discussed which may explain these observations.

Localization of Branching Enzyme in Potato Tuber Cells with the Use of Immunoelectron Microscopy

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.

Mixed mono- and multilayers of poly(isocyanide)s with non-linear optically active side chains☆

Abstract The properties and structure of Langmuir-Blodgett mono- and multilayers of several poly(isocyanide)s with non-linear optically active side-chains were studied. These polymers formed very rigid layers or layers which appeared to be unstable. To circumvent this problem they were mixed with other poly(isocyanide)s or with amylose-butyrate. Transmission electron microscopy studies of the polymeric mixtures revealed that phase separation occured in all cases and that only poly(isocyanide)s substituted with hydrophilic side chains formed monomolecular layers. Depending on the nature of the polymer which was used for mixing, the dye polymers could be deposited with Y- or Z-type transfer. Second-harmonic intensities generated from these films were small in the case of the Y-type multilayered films and moderately high in the case of the Z-type films.

SCANNING-TRANSMISSION ELECTRON-MICROSCOPY OF BIOLOGICAL MACROMOLECULES

The practical usefulness of a STEM (Scanning Transmission Electron Microscope) for the study of the structure of biological macromolecules has been investigated using a STEM attachment connected to a TEM (Transmission Electron Microscope), which in one case was equipped with a tungsten hairpin cathode, and in the other case with a field emission gun. The point to point resolution has been determined. Results obtained in STEM dark field from light negatively stained specimens are compared with results obtained in TEM bright field from normal negatively stained specimens. In addition unstained molecules have been visualized. Some remarks are made about preparation methods suitable for STEM.

A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPFlong. The N-terminal domain of HPFlong binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPFlong binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPFlong, and the Shine–Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPFlong due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPFlong-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria.When bacteria enter the stationary growth phase, protein translation is suppressed via the dimerization of 70S ribosomes into inactive complexes. Here the authors provide a structural basis for how the dual domain hibernation promotion factor promotes ribosome dimerization and hibernation in bacteria.

Cryo-EM structure of a tetrameric cyanobacterial photosystem I complex reveals novel subunit interactions

Photosystem I (PSI) of the thermophilic cyanobacterium Chroococcidiopsis sp. TS-821 (TS-821) forms tetramers Li et al. (2014). Two-dimensional maps obtained by single particle electron microscopy (EM) clearly show that the tetramer lacks four-fold symmetry and is actually composed of a dimer of dimers with C2 symmetry. The resolution of these negative stain 2D maps did not permit the placement of most of the small PSI subunits, except for PsaL. Therefore cryo-EM was used for 3D reconstruction of the PSI tetramer complex. A 3D model at ~11.5Å resolution was obtained and a 2D map within the membrane plane of ~6.1Å. This data was used to build a model that was compared with the high-resolution structure of the PSI of Thermosynechococcus elongatus (T. elongatus) at 2.5Å. This comparison reveals key differences in which subunits are involved in the two different interfaces, interface type 1 within a dimer and interface type 2 between dimers. The type 1 interface in TS-821 is similar to the monomer interface in the trimeric PSI from T. elongatus, with interactions between subunits PsaA, -B, -I, -L and M. In type 2 the interaction is only between PsaA, -B and -L. Unlike the trimeric PSI, the central cavity of the complex is not filled with the PsaL-derived helical bundle, but instead seems filled with lipids. The physiological or evolutionary advantage of the tetramer is unknown. However, the presence of both dimers and tetramers in the thylakoid membrane suggest a dynamic equilibrium that shifts towards the tetramers in high light.

MONOLAYERS AND LANGMUIR-BLODGETT MULTILAYER FILMS OF A CONJUGATED AZO POLYMER

A black pi-conjugated azo polymer was synthesized by oxidative coupling of 3,5-diamino-1-octadecylbenzoate. The polymer, with a number average molecular weight of about 16 000, was soluble in chloroform. Monolayer formation was studied by transmission electron microscopy and the structure of the deposited Langmuir-Blodgett multilayer film was investigated with small angle X-ray diffraction and Fourier transform IR spectroscopy. A smooth monolayer was obtained when, after spreading, the material was allowed to disintegrate without any applied surface pressure for 18 h at 20-degrees-C and 1 h at 40-degrees-C. Monolayers could be transferred successfully onto different substrates at high temperature (40-degrees-C) and high pressure (30 mN m-1). The deposition was of the Y type with transfer ratios of 1 on both the downstroke and the upstroke. It was concluded that the aliphatic side chains are not able to crystallize and therefore form amorphous layers.

Biosynthesis of starch; identification of potato starch enzymes

Abstract Two important starch enzymes, granule-bound starch synthase and branching enzyme, were purified from potato tubers and characterized by immunological comparison with the corresponding enzymes of other plants. Granule-bound starch synthase was identified as a 60-kd protein homologous to the corresponding enzymes of maize and amaranth; the enzyme was missing in amylose-free potato starch granules. Branching enzyme of potato tubers was purified as a single protein species of 79 kd which appeared to be homologous to maize branching enzyme I, but much less to branching enzymes IIa and IIb.