Protein crystallization successful

In this chapter we describe the basic principles involved in the controlled production and modification of two-dimensional protein crystals. These are synthesized in nature as the outermost cell surface layer (S-layer) of prokaryotic organisms and have been successfully applied as basic building blocks in a biomolecular construction kit. Most importantly, the constituent subunits of the S-layer lattices have the capability to recrystallize into iso-porous closed monolayers in suspension, at liquid-surface interfaces, on lipid films, on liposomes, and on solid supports (e.g., silicon wafers, metals, and polymers). The self-assembled monomolecular lattices have been utilized for the immobilization of functional biomolecules in an ordered fashion and for their controlled confinement in defined areas of nanometer dimension. Thus, S-layers fulfill key requirements for the development of new supramolecular materials and enable the design of a broad spectrum of nanoscale devices, as required in molecular nanotechnology, nanobiotechnology, and biomimetics [1-3]. 

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

Weber, P. C. (1990). A protein crystallization strategy using automated grid searches on successively finer grids. 

Table 17.1 lists a number of successful drugs on the market that were designed using knowledge and analysis of protein crystal structures. Note that the list is dominated by HIV protease inhibitors, drugs for the treatment of AIDS. The speed by which these... 

The software required for the quantitative evaluation and wavelength normalisation of Laue data has been successfully developed at Daresbury since the feasibility of recording full white beam Laue patterns from protein crystals was established. The software and the method have been tested using Laue data from crystals of pea lectin. As an example of the statistical quality (to 3 A resolution) the mean fractional change on F between monochromatic and Laue pea lectin data was 11 % and the same quantity between conventional source monochromatic and SR monochromatic pea lectin data is 8% (Helliwell et al., 1986). 

A knowledge of the structure of proteins has long been recognized as fundamental to an understanding of their chemical and biological functions. One technique for the successful determination of the X-ray crystal structure of proteins was initiated by Perutz in 1954 and involved the isomorphous replacement of heavy atoms into the protein crystal.412 The development of this technique was recently reviewed.413... 

Early protein crystallographers, proceeding by analogy with studies of other crystalline substances, examined dried protein crystals and obtained no diffraction patterns. Thus X-ray diffraction did not appear to be a promising tool for analyzing proteins. In 1934, J. D. Bernal and Dorothy Crowfoot (later Hodgkin) measured diffraction from pepsin crystals still in the mother liquor. Bernal and Crowfoot recorded sharp diffraction patterns, with reflections out to distances in reciprocal space that correspond in real space to the distances between atoms. The announcement of their success was, in effect, a birth announcement for protein crystallography. 

The ultimate in structural studies would, of course, involve X-ray crystallographic studies of protein crystals prepared from nonaqueous solvents of the kind that are now being so successfully carried out with certain protein crystals prepared from aqueous media (Kendrew et al., 1961). A priori, there is no reason to exclude the possibility that proteins might be crystallized from pure nonaqueous solvents, although no reports of such attempts have appeared. This is particularly so in view of the fact that in certain pure solvents, proteins appear to exhibit a more highly ordered (helical) conformation than they do in water solution. 

Lipid cubic (51) and sponge (52) phases, as well as bicelles (53), are alternatives to detergents that have been applied successfully to membrane protein crystallization. In these instances, the protein is embedded in a lipid bilayer environment, which is considered more natural compared with the detergents that form micellar phases. In the recent high-resolution crystal structure of the human 32 adrenergic G-protein-coupled receptor, lipid cubic phase was used with necessary cholesterol and 1,4-butandiol additives (54). The cholesterol and lipid molecules were important in facilitating protein-protein contacts in the crystal. 

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