Prokaryotic systems, protein structure-function studies

In addition to the in vitro studies of well-defined systems that have been discussed here, NMR spectroscopy can also be applied to living systems or complex substance mixtures (146). This broad applicability is an advantage of NMR spectroscopy over X-ray crystallography. Early in vivo NMR studies that mostly identify small metabolite molecules by P or H ID experiments have led to different applications. In-cell NMR uses isotopic labeling combined with two or higher dimensional NMR experiments for structural studies of the phosphorylation state of a given protein in the cellular environment or of intrinsically unstructured proteins. In-cell NMR applications in prokaryotic systems cover structural and functional studies (i.e., protein-protein interactions, protein dynamics, automated structure determination, and de novo resonance assignments (147-149)). 

Copper ion homeostasis in prokaryotes involves Cu ion efflux and sequestration. The proteins involved in these processes are regulated in their biosynthesis by the cellular Cu ion status. The best studied bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae. Cellular Cu levels in this bacterium control the expression of two P-type ATPases critical for Cu homeostasis (Odermatt and Solioz, 1995). The CopA ATPase functions in Cu ion uptake, whereas the CopB ATPase is a Cu(I) efflux pump (Solioz and Odermatt, 1995). The biosynthesis of both ATPases is regulated by a Cu-responsive transcription factor, CopY (Harrison et al., 2000). In low ambient Cu levels Cop Y represses transcription of the two ATPase genes. On exposure to Cu(I), CopY dissociates from promoter/operator sites on DNA with a for Cu of 20 jlM (Strausak and Solioz, 1997). Transcription of copA and copB proceeds after dissociation of CuCopY. The only other metal ions that induce CopY dissociation from DNA in vitro are Ag(I) and Cd(II), although the in vivo activation of copA and copB is specihc to Cu salts. The CuCopY complex is dimeric with two Cu(I) ions binding per monomer (C. T. Dameron, personal communication). The structural basis for the Cu-induced dissociation of CopY is unknown. Curiously, CopY is also activated in Cu-dehcient cells, but the mechanism is distinct from the described Cu-induced dissociation from DNA (Wunderh-Ye and Solioz, 1999). 

The SH3 fold has been observed in other protein domains with distinct function (relevant information can be found in reference 37). Recently, a group of bacterial proteins bearing significant sequence similarity to the SH3 domain was discovered (38). Whether these prokaryotic domains share the SH3 fold and are involved in PPII recognition is currently unknown. The simplicity of the fold, the great wealth of structural information, and the ease of its biochemical handling have made the SH3 domain a very-well-characterized model system for the study of proteinfolding mechanisms (see for instance references 39-46), but these studies will not be reviewed here. 

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