Protein crystallization effectors

Since these proteins are difficult to crystallize and site-directed mutagenesis has certain limitations, PAL can find much use in exploring pathways, transducers, and effectors in signal transduction as well as some of their main structural features. 

Saeo, M.K., Moure, C.M., Burnett, J.C., Joshi, G.S., and Abraham, D.J. High-resolution crystal structure of deoxy hemoglobin complexed with a potent allosteric effector. Protein Sci. 2001, 10, 951-957. 

But where there is an equilibrium among two or more conformations of the enzyme in solution, crystallization may select out only one of the conformations. a-Chymotrypsin has a substantial fraction of an inactive conformation present under the conditions of crystallization, but only the active form of the enzyme crystallizes. An allosteric effector molecule that changes the conformation of the protein in solution may have no effect on the crystalline protein, as, for example, with phosphorylase b.5A The enzyme is frozen in one conformation, with the crystal lattice forces preventing any conformational change. On the other hand, the addition of an effector to phosphorylase a causes the crystals first to crack and then to anneal, giving crystals of the enzyme in a second conformation. 

Just as the auto mechanic sometimes has parts left over, electron-density maps occasionally show clear, empty density after all known contents of the crystal have been located. Apparent density can appear as an artifact of missing Fourier terms, but this density disappears when a more complete set of data is obtained. Among the possible explanations for density that is not artifactual are ions like phosphate and sulfate from the mother liquor reagents like mercaptoethanol, dithiothreitol, or detergents used in purification or crystallization or cofactors, inhibitors, allosteric effectors, or other small molecules that survived the protein purification. Later discovery of previously unknown but important ligands has sometimes resulted in subsequent interpretation of empty density. 

We chose H-RAS as the first target protein for several reasons. RAS proteins are, perhaps, the most intensively studied and best understood signal transduction proteins from the perspective of structure and function [8], The H-RAS crystal structure has been determined to the level of 1.5 A, and the domains involved in guanine nucleotide binding and hydrolysis, effector interactions, interactions with regulating proteins, and processing have been extensively characterized. RAS proteins are initially synthesized in the cytoplasm where they undergo a series of post-translational modifications at their carboxy-terminal sequence, the CaaX-box (where C is cysteine, a an aliphatic amino acid, and X either serine or... 

As already noted, temperature may be of crucial importance or it may have little bearing at all. In general, it is wise to duplicate crystallization trials and conduct parallel investigations at 4°C and at 25°C. Even if no crystals are observed at either temperature, differences in the solubility behavior of the protein with different precipitants and with various effector molecules, may give some indication as to whether temperature is likely to play an important role. If crystals are observed to grow at one temperature and not, under otherwise identical conditions, at the another, then further refinement of this variable may be justified. This is accomplished by conducting the trials under the previously successful conditions over a range of temperatures centered on the one that initially yielded crystals. 

Initially, a pH range of 3.5 to 9.0 should be explored in intervals of 0.5, but the range should be extended, abridged, or modified as appropriate. Generally, it is sufficient to set up two parallel sets of trials and maintain one set at 4°C and the other at 25°C. This will provide an indication of the possible influence and value of temperature as a variable. If crystals of any sort are obtained in the first round of trials, then the coarse matrix of conditions is more finely sampled, evaluated, and in successive rounds, the growth of the crystals optimized. If no crystals are obtained, ligand complexes or alternative forms of the protein are explored. If this fails, then effectors such as metal ions and detergents are introduced, and so on. 

The best-characterized class I RNR is that of . coli. Excellent reviews have recently been written on this enzyme (37-39, 73). The enzyme consists of the nonidentical proteins R1 and R2, each of which is a homodimer. The holoenzyme therefore has an a2 2 structure. The crystal structures of the E. coli R1 and R2 proteins have recently been reported (18, 19, 74). Protein R1 has 2 x 761 residues and contains the substrate binding sites, as well as the binding sites for the allosteric effectors. The exact localization of the specific binding sites for the effectors and the substrate, as well as the area of subunit interaction in the active enzyme, has so far only been modeled into the R1 structure. Protein R2 has 2 x 375 residues and contains the sites for the iron center as well as the tyrosyl radical. The crystal structure is obtained for the met form, i.e., without tyrosyl free radical. There is one site for... 

The switch function of the a-subunit of the heterotrimeric G proteins is founded on the change between an active Ga-GTP conformation and an inactive Ga-GDP conformation. In this process, interaction sites with downstream effector proteins are exposed that are not available in the inactive GDP-state. The structural difference between the two conformations was explained for the transducin, Ga t, by crystallization and structural characterization of the inactive GDP form and the active GTPyS form (Lambright et al., 1994). The structures of both forms of Ga t are shown in Fig. 5.21. 

The determination of the specificity of interaction of G-proteins with receptor subtypes, as weU as with different effectors, remains to be investigated. Other important questions are how in molecular terms the interactions result in structural changes, and how these in turn cause activity changes. Progress in analyzing the crystal structure of transducin complexed with its activators (Noel et al., 1993 Lambright et ah, 1994 Sondek et al, 1994) raises hopes that answers to these questions will also be obtained for the smooth muscle G-proteins. 

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