Jens Dietrich ; Catherine Venien-Bryan. Publisher: Imperial College Press , This specific ISBN edition is currently not available. View all copies of this ISBN edition:. Synopsis Strategies for Two-Dimensional Crystallization of Proteins Using Lipid Monolayers presents an overview of different methods that lead to structure determination by electron microscopy. Buy New Learn more about this copy. About AbeBooks. Customers who bought this item also bought. Stock Image.
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Published by Imperial College Press New Quantity Available: Seller Rating:. Published by Imperial College Press , London New Hardcover Quantity Available: 1. Published by Imperial College Press. New Hardcover Quantity Available: 5. New Hardcover Quantity Available: Although it has been shown for several enzymes that they retain their activity after immobilization see Refs.
Not only the immobilization might affect the activity, also the mere presence of His tags can, in principle, have an influence. Obviously, the presence of His tags on the proteasomal exterior has no strong effects on peptide hydrolysis. In the crystal structure of the T. However, because N-terminal residues 1 to 12 were disordered in the case of the Thermoplasma proteasome, it is likely that here too the channel is partially occluded. Surface plasmon resonance measurements allowed the authors of Ref. In the experiments described in this article we have measured the activity of immobilized proteasomes by direct observation of the products of hydrolysis.
However, several factors may cause this more pronounced reduction of activity compared with proteasomes bound to vesicles. Clearly, access to the entrance channels of proteasomes will be partly blocked at such high coverage. Additionally, the substrate concentration at the interface will be different for a flat, extended bilayer and for small vesicles for geometric reasons.
One could envisage that the channel directed to the lipid layer is less accessible than the opposite one and a quantification of the activity of proteasomes in this orientation could provide some insight into the functional interplay of both channels.
Structure - Approaches
This observation is consistent with a scenario in which, at least in the case of small substrates, both substrates and products can pass through the same pore. However, it is also possible that the entrance facing the lipid film is not obstructed sufficiently. Complete sealing of this entrance has to be expected for two-dimensional crystals on lipid films, but the corresponding measurements of activity are very problematic because the percentage of two-dimensional crystals in the preparation cannot be quantified accurately enough.
AFM allows one to visualize biological structures in action under near physiological conditions and, in favorable cases, with subnanometer resolution In many cases, closely packed arrays or two-dimensional crystals have been found to facilitate attaining high resolution One strategy that has been explored to obtain such arrays is the use of streptavidin films grown on biotinylated lipid films 42 as a matrix for growing oriented arrays of biotinylated proteins 43 , However, the self-organization of the underlying two-dimensional crystalline streptavidin matrix imposes severe steric limitations on the two-dimensional arrangement of proteins bound to it and, moreover, these proteins need to be biotinylated in a site-specific manner.
Similar restrictions apply to self-assembled monolayers carrying NTA groups 45 ; nevertheless, they have been used successfully to monitor the transcription of circular, single-stranded DNA templates by AFM First, proteins are frequently expressed with a His tag in the first instance to facilitate their purification. Second, lipid monolayers are more flexible as a matrix than self-assembled monolayers; they allow one to influence the lateral organization via the phase behavior of the chosen lipid.
If desired, a condensed lipid film will keep immobilized molecules separated, whereas a fluid lipid film will allow lateral protein-protein interactions and may thus favor dense arrays or even two-dimensional crystallization. The appearance of the proteasomes in the crystals did not change with time during repeated scanning.
Based on AFM recordings, yeast 20 S proteasomes have been reported to switch between 2 distinct conformations In contrast, the AFM images of proteasomes in two-dimensional arrays did not reveal the existence of different conformational states. The AFM images gave insight into the vertical packing of crystalline proteasomes, which cannot be deduced from two-dimensional electron micrographs.
Proteasomes were found to be arranged in 2 levels in AFM topographs. Apparently, proteasomes within the two-dimensional crystals optimize their packing not only by interlocking laterally, but also by a vertical shift along their cylinder axis. As a result, one ring of the proteasome fits into its neighbors waist. To pack in that manner, proteasomes have to be able to move away from the interface by 2 nm.
Assuming that all proteasomes within a crystal are actually bound to the lipid film requires that their linkage be rather flexible. Such a flexible linker might prove useful in many systems and could similarly be realized in the form of a short stretch of disordered residues leading up to a His tag. In addition to facilitating high resolution studies, immobilizing proteins in a predetermined orientation will also enable dynamic studies of their interaction with substrates or cofactors in real time. However, the orientation of unspecifically adsorbed molecules will often be random or unfavorable.
In contrast, the His tag-nickel-NTA system allows one to bind molecules in an orientation conducive to further investigation. For example, the interaction of regulatory particles with proteasomes bound end-on will be readily accessible to an AFM tip as height fluctuations. We gratefully acknowledge E. Voges for sharing molecular biology expertise, and M. Boicu for sequencing.
We thank C. Zeth for assistance with the rendering software. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. Hutschenreiter and R. You'll be in good company.
Journal of Lipid Research. Figure 1 Schematic representation of the T. Previous Section Next Section.
(IUCr) Femtosecond X-ray diffraction from two-dimensional protein crystals
Activity of Proteasomes Immobilized at Nickel-chelating Lipid Interfaces The activity of proteasomes was tested after immobilization on two kinds of bilayer specimens, that is on vesicle bilayers and on mica supported bilayers. Electron Microscopy To investigate the interaction of proteasomes with lipid films, 1. Atomic Force Microscopy To obtain AFM atomic force microscope topographs of the lipid-proteasome assembly, His-tagged proteasomes were immobilized on supported nickel-chelating lipid bilayers.
Proteasomes His-tagged in Various Positions Can be Efficiently Expressed and Purified The main goal of this study was to immobilize the cylindrical-shaped 20 S proteasome with predetermined orientation, end-on and side-on. View this table: In this window In a new window. Table I Degradation of casein by proteasomes with and without His-tag nmol degraded per h and mg of proteasome.
Proteasomes Can Be Oriented at Metal-chelating Lipid Interfaces via the Location of His Tags To test whether site-specific binding between His-tagged proteasomes and nickel-chelating interfaces would permit one to control orientation, proteasomes were allowed to interact with nickel-chelating lipid layers. Figure 2 Electron micrograph of cylindrical wild-type 20 S proteasomes adsorbed on carbon film. Figure 3 According to electron micrographs the orientation of His-tagged proteasomes bound to metal-chelating lipid films depends on the location of the His tags and is highly uniform.
Femtosecond X-ray diffraction from two-dimensional protein crystals
The Activity of Proteasomes Bound to Metal-chelating Lipid Interfaces Is Well Preserved To demonstrate that immobilization of His-tagged proteins at metal-chelating lipid interfaces is biocompatible, the activity of immobilized proteasomes was compared with the activity of proteasomes in solution. Uniform Orientation of His-tagged Proteasomes on Nickel-chelating Lipid Films Promotes Their Two-dimensional Crystallization Incubation of His-tagged proteasomes with fluid metal-chelating lipid films for longer periods of time often resulted in two-dimensional crystalline arrays see Fig.
Binding of His-tagged Proteins to Metal-chelating Lipid Interfaces Is Biocompatible Although it has been shown for several enzymes that they retain their activity after immobilization see Refs. Supported Chelating Lipid Bilayers Are a Versatile Support for Scanning Probe Microscopy of Proteins AFM allows one to visualize biological structures in action under near physiological conditions and, in favorable cases, with subnanometer resolution Previous Section.
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CrossRef Medline Google Scholar. Dietrich C. Dorn I. Coux O. Baumeister W. Voges D. Wenzel T. Lupas A. Zwickl P. Cell Biol. Schmidt M. These methods require small volume of low concentration of proteins, making it possible to explore more conditions with the same amount of protein. These automated methods outperform traditional 2D crystallization approaches in terms of accuracy, flexibility, and throughput.
Membrane protein, Lipid, Structural biology, Detergent, Lipid monolayer, Reconstitution in lipid bilayer.
Antibody Data Search Beta. Authors: Philippe Rosier 1 ,. Nicolas Dumesnil 1 ,. Gauthier Esnot 1. Philippe Rosier 1 ,.
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