Intermediate-compact forms in protein folding and translocation

This work discusses the significance of intermediate compact forms (ICF) in protein structure, folding and translocation.; The role of hydration and volume changes measured by dilatometry was investigated with lysozyme and lactalbumin. The volume change results of hydrating dry lysozyme were large and negative indicating a contribution of the structuring of water. The refolding of denatured lysozyme produces a positive volume change which may reflect the initial hydrophobic collapse consequent to refolding. A slow volume change observed on the refolding of lysozyme was further characterized using ultraviolet, circular dichroism and fluorescence spectroscopy. This was indicative of a plausible ICE being populated as a kinetic intermediate. The refolding of lactalbumin from molten globule to native state by pH titration produces {dollar}-{dollar}119 ml/mole volume change confirming the expanded state of the protein at acid pH. Lysozyme exhibits little change under similar conditions. The difference observed between these two homologous proteins reflects their conformational properties emphasizing the importance of 'context' in protein structures.; The plausible involvement of ICFs in vivo translocation was investigated for the hybrid protein pro-OmpA nuclease (OmpA signal peptide fused to staphylococcal nuclease) by biophysical characterization of the purified precursor. The secondary structure content of the pro-OmpA nuclease by CD is similar to the mature nuclease. Characterization of the tertiary structure by near-UV CD shows a significant difference in ellipticity of the aromatic groups in the pro-OmpA nuclease.; To further elucidate differences in tertiary structure, thermal denaturation of the two proteins was performed using CD and fluorescence. In the presence of the OmpA signal peptide, the mature nuclease ceases to follow a two-state transition on heat denaturation. Using dichroism at far-UV wavelengths, no significant difference in T{dollar}sb{lcub}rm m{rcub}{dollar} was observed between pro-OmpA and the mature nuclease, but a marked difference was evident from heat denaturation studies by fluorescence. These studies confirm that the tertiary structure around the tryptophan is disturbed in the presence of the signal peptide. The perturbation of the tertiary structure observed for a protein with a signal peptide may be pertinent for efficient translocation.