15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.

Biochemical and biophysical characterization of refolded Drosophila DPP, a homolog of bone morphogenetic proteins 2 and 4.

The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15 degreesC). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coli-expressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250-400 amino acids) N-terminal prodomains of transforming growth factor-beta superfamily members, shown to act as intramolecular chaperones in vivo.