No abstract

The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. PPII helices play vital roles in a variety of processes including signal transduction, transcription, and cell motility. Proline-rich regions of sequence are often assumed to adopt this structure. Remarkably, little is known about the physical determinants of this secondary structure type. In this study, we have explored the formation of PPII helices by a short poly(proline) peptide. In addition, the results from experiments used to determine the propensities for apolar residues, plus glycine, asparagine, and glutamine, to adopt this structure in a poly(proline)-based host peptide are reported here. Proline possesses the highest intrinsic propensity, with glutamine, alanine, and glycine having surprisingly high propensities.-Branched residues possess the lowest propensities of the residues examined. It is postulated that propensities possessed by apolar residues are due in part to peptide-solvent interactions, and that the remarkably high propensity possessed by glutamine may be due to a side chain to backbone hydrogen bond. These data are the first step toward a molecular understanding of the formation of this important, and yet little studied, secondary structure.In recent years, the left-handed polyproline II (PPII) 1 helical conformation has been elevated from the status of a relatively rare and seemingly uninteresting secondary structure to one that is surprisingly common and of the utmost importance. This structure plays a central role in numerous vital processes including signal transduction, transcription, cell motility, and the immune response. Proline-rich ligands of the cytoskeletal protein profilin (1), as well as those of the SH3, WW, and EVH1 protein interaction domains, are bound in this conformation (2). The peptide ligands of class II MHC molecules are also bound in the PPII conformation (3). PPII helices are major features of collagens (4) and plant cell wall proteins (5). The PPII helix is believed to be the dominant conformation for many proline-rich regions of sequence (PRRs) (6). Sequences not rich in proline also adopt this structure. For example, poly(lysine), poly(glutamate), and poly(aspartate) peptides form PPII helices (7). Around 2% of all residues in known protein structures are found in PPII helices at least four residues long (8, 9). As many as 10% of all residues are found in the PPII conformation, although not necessarily as part of PPII helices (10). PPII helices have also been hypothesized to be a major component of protein denatured states (11-14), giving them a role in a most fundamental process. Recently, Blanch et al. (15) have suggested that the PPII helix might be the precursor conformation in amyloid formation. Given the preceding, it is truly remarkable how little is known about the physical determinants of the PPII helical conformat...

Superoxide dismutases (SODs) catalyze the de-activation of superoxide. SODs therefore acquired great importance as O2 became prevalent following the evolution of oxygenic photosynthesis. Thus the three forms of SOD provide intriguing insights into the evolution of the organisms and organelles that carry them today. Although ancient organisms employed Fe-dependent SODs, oxidation of the environment made Fe less bio-available, and more dangerous. Indeed, modern lineages make greater use of homologous Mn-dependent SODs. Our studies on the Fe-substituted MnSOD of Escherichia coli, as well as redox tuning in the FeSOD of E. coli shed light on how evolution accommodated differences between Fe and Mn that could affect SOD performance, in SOD proteins whose activity is specific to one or other metal ion.

We propose that the apparent catalytic inactivity of Mn- and Fe-substituted superoxide dismutases (SODs) reflects E°s that are either lower (Fe-sub-(Mn)SOD) or higher (Mn-sub-(Fe)SOD) than those of native Fe- or Mn-SODs. In support, we show that the E° of Fe-sub-(Mn)SOD (Fe substituted into Mn-SOD protein) is −240 mV vs NHE, almost 0.5 V lower than our E° of 220 mV for Fe-SOD. The E° of Fe-sub-(Mn)SOD is lower than that of O2/O2 •- and therefore is sufficient to explain Fe-sub-(Mn)SOD's inactivity. Indeed, Fe-sub-(Mn)SOD is shown to be unable to oxidize O2 •-. Alternate causes of inactivity are ruled out by our demonstration that Fe-sub-(Mn)SOD retains the ability to reduce O2 •-. Thus, the active site remains active with respect to substrate binding and proton and electron transfer. Finally, we show that Fe-sub-(Mn)SOD's inactivity with respect to O2 •- oxidation cannot be solely due to competitive inhibition by OH-. Thus, our proposal provides a simple chemical basis for the observed catalytic inactivity of metal-exchanged Mn- or Fe-SODs and suggests that these strongly homologous enzymes may provide important insights into mechanisms of redox midpoint potential tuning in proteins.