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Searching for silver bullets: an alternative strategy for crystallizing macromolecules.

Published on Dec 1, 2006in Journal of Structural Biology3.754
· DOI :10.1016/j.jsb.2006.09.006
Alexander McPherson51
Estimated H-index: 51
(UCI: University of California, Irvine),
Bob Cudney4
Estimated H-index: 4
Abstract
Abstract Based on a hypothesis that various small molecules might establish stabilizing, intermolecular, non covalent crosslinks in protein crystals and thereby promote lattice formation, we carried out three separate experiments. We assessed the impact of 200 chemicals on the propensity of 81 different proteins and viruses to crystallize. The experiments were comprised of 18 240 vapor diffusion trials. A salient feature of the experiments was that, aside from the inclusion of the reagent mixes, only two fundamental crystallization conditions were used, 30% PEG 3350, and 50% Tacsimate™ at pH 7. Overall, 65 proteins (85%) were crystallized. Most significant was that 35 of the 65 (54%) crystallized only in the presence of one or more reagent mixes, but not in control samples lacking any additives. Among the most promising types of reagent mixes were those composed of polyvalent, charged groups, such as di and tri carboxylic acids, diamino compounds, molecules bearing one or more sulfonyl or phosphate groups, and a broad range of common biochemicals, coenzymes, biological effectors, and ligands. We propose that an alternate approach to crystallizing proteins might be developed, which employs a limited set of fundamental crystallization conditions combined with a broad screen of potentially useful small molecule additives.
  • References (43)
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References43
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#1Alexander McPherson (Penn State Milton S. Hershey Medical Center)H-Index: 4
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The determination of high-resolution structures of proteins requires crystals of suitable quality. Because of the new impetus given to structural biology by structural genomics/proteomics, the problem of crystallizing proteins is becoming increasingly acute. There is therefore an urgent requirement for the development of new efficient methods to aid crystal growth. Nucleation is the crucial step that determines the entire crystallization process. Hence, the holy grail is to design a “universal n...
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#1Peter Nollert (deCODE genetics)H-Index: 21
Abstract This review provides detailed procedures for the crystallization of membrane proteins via the lipidic cubic phase method. Bacteriorhodopsin-specific, hands-on protocols are given for (i) the preparation of bacteriohordopsin from purple membrane by monomerization in octylglucoside and gel filtration chromatography or by selective extraction after pre-treatment with dodecyl-trimethylammonium bromide, (ii) the incorporation of bacteriorhodopsin into lipidic cubic phases by mixing in vials ...
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#1D.W. Bolen (UTMB: University of Texas Medical Branch)H-Index: 1
Protein solubility and stability are issues of consideration in attempts to crystallize proteins. These two properties of proteins are also at issue in the cells of organisms that have adapted to water stress conditions that could ordinarily denature or inactivate some proteins. Most organisms that have adapted to environmental stresses have done so by production and accumulation of certain small organic molecules, known as osmolytes, that arose by natural selection and have the ability to stabi...
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#1Michael C. Wiener (UVA: University of Virginia)H-Index: 23
Membrane protein structural biology is a frontier area of modern biomedical research. Twenty to thirty-five percent of the proteins encoded by an organism's genome are integral membrane proteins. Integral membrane proteins, such as channels, transporters, and receptors, are critical components of many fundamental biological processes. Also, many integral membrane proteins are important in biomedical and biotechnological applications; the majority of drug targets are integral membrane proteins. T...
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#1Kim D. Collins (UMB: University of Maryland, Baltimore)H-Index: 6
Abstract Sephadex G-10 gel sieving chromatography, Jones-Dole viscosity B coefficients, and solution neutron and X-ray diffraction are used to show that small ions of high charge density (e.g., sulfate, phosphate, the carboxylate, sodium, and fluoride) are strongly hydrated (kosmotropes) whereas large monovalent ions of low charge density (e.g., ammonium, chloride, potassium, and the positively charged amino acid side chains) are weakly hydrated (chaotropes). The heats of solution of the crystal...
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Abstract The need for high-resolution structure information on membrane proteins is immediate and growing. Currently, the only reliable way to get it is crystallographically. The rate-limiting step from protein to structure is crystal production. An overview of the current ideas and experimental approaches prevailing in the area of membrane protein crystallization is presented. The long-established surfactant-based method has been reviewed extensively and is not examined in detail here. The focu...
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Abstract A method is described to identify small molecule ligands that stabilize proteins. The procedure is based on the hypothesis that molecules of various sizes containing two to four charges should occasionally bind to unpaired charged sites on the surface of proteins and by crosslinking such residues stabilize the native state of the liganded protein. A simple turbidity assay is employed that detects inhibition of protein aggregation under selected sets of conditions. Eight test proteins we...
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