Yeast protein crystallization

Electrophoresis of a crystalline preparation revealed one major enzymatically active component and a minor inactive one (Negelein and Wulff, 1937). The smaller, inactive constituent varied from 5% to 20% of the total protein. Re-examination of the electrophoretic properties of yeast ADH crystals confirmed the presence of one slow, presumably active, and one fast-moving, presumably inactive component. Their relative amounts did not change systematically with recrystallization as shown by area analysis. The percentage of inactive component was a function of age of the solution and duration of preliminary dialysis. Preparations dialyzed for 20 hours at pH 5 contained as much as 14 % to 25 % of the inactive component, while dialysis for 4 hours showed as little as 6 %. The second component was assumed to be an inactive transformation product of the active enzyme (Hayes and Velick, 1954). 

Page 1176 (Figure 28 11) is adapted from crystallographic coordinates deposited with the Protein Data Bank PDB ID 6TNA Sussman J L Holbrook S R Warrant R W Church G M Kim S H Crystal Structure of Yeast Phenylalanine tRNA I Crystallographic Refinement / Mol Biol 1978 126 607 (1978)... 

Figure 9.3 Comparison of the consensus nucleotide sequence of the TATA box (a) and the sequences of the DNA fragments used in the crystal structure determinations of the TATA box-binding proteins from yeast (b) and the plant Arabidopsis thaliana (c).

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

Umland, T. C., Taylor, K. L., Rhee, S., Wickner, R. B., and Davies, D. R. (2001). The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p. Proc. Natl. Acad. Sci. USA 98, 1459-1464. 

Proteasome inhibitors have been instrumental in identifying numerous protein substrates and in elucidating the importance of the proteasome/ubiquitin pathway in many biological processes. Initially, non-specific cell-penetrating peptide aldehydes were used for this purpose. More recently, it became possible to synthesize compounds with increased potency and selectivity (Adams et al. 1998 Elofsson et al. 1999). Furthermore, based on the crystal structure of the yeast and bovine liver CP (Groll et al. 1997 Unno et al. 2002), molecular modeling can now be used to engineer improved inhibitors. 

---