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Additional file 1. Supplementary information.

The crystal structure of the vitamin B(6)-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 A resolution. The model was refined to an R-factor of 11.7% (R(free) = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from Escherichia coli and to that of phosphoserine aminotransferase from a facultative alkaliphile, Bacillus circulans subsp. alkalophilus. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5'-phosphate are replaced, and an additional hydrogen bond to the O3' atom of the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent-accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in the Bacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme from Escherichia coli contains two additional beta-strands in a surface loop with a third beta-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase.

The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable VH domain, (Gly4Ser)3 linker peptide, and light chain variable VL domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a V8- fold decrease in the affinity (Ka = 5.1U10 8 M 31 in RIA and 1.8U U10 8 M 31 in ELISA) vs. the parent IgG2a/U U monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydro- phobic probe 8-anilino-1-naphthalenesulfonate (ANS) recogniz- ing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding. z 1998 Federation of European Biochemical Societies.

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5′-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5′-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3°C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes. Proteins 2006. © 2006 Wiley-Liss, Inc.

Additional file 1. Supplementary information.