Experimental and theoretical analyses of the domain architecture of mammalian protein disulphide-isomerase.

The high resolution structure of full-length protein disulphide-isomerase (PDI) has not been determined, but the polypeptide is generally assumed to comprise a series of consecutive domains. Models of its domain organisation have been proposed on the basis of various sequence-based criteria and, more recently, from structural studies on recombinant fragments corresponding to putative domains. We here describe direct studies of the domain architecture of full-length mammalian PDI based on limited proteolysis of the native enzyme. The results are consistent with an emerging model based on the existence of 4 consecutive domains each with the thioredoxin fold. The model was further tested by expressing recombinant fragments corresponding to alternative domain models and to truncated domains; the observed properties of these purified fragments supported the 4-domain model. A multiple alignment of many PDI-like sequences was generated to test whether domain boundaries could be predicted from any features of the alignment, such as sequence variability or hydrophilicity; neither of these parameters reliably predicted the domain boundaries determined by experiment.

The folding and assembly pathway of tumour necrosis factor TNF alpha, a globular trimeric protein.

The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor-alpha (TNF) were studied by chemical cross-linking. This revealed the rapid accumulation of a dimeric intermediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8-anilino-1-naphthalene sulfonic acid fluorescence are first order and, combined with the kinetics of association, reveal the presence of folding steps both before and subsequent to formation of the trimer. Results from gel exclusion chromatography and kinetics, together with the existence of an acid-induced molten globule, support the conclusion that TNF folds and assembles through a trimeric molten globule.

Binding of defined regions of a polypeptide to GroEL and its implications for chaperonin-mediated protein folding.

Unfolded rhodanese in a complex with the chaperonin GroEL was subjected to limited proteolysis. Sequence analysis indentified a GroEL-bound fragment of approximately 11,000 M(r) and a well defined fragment of approximately 7,000 M(r) from the two homologous domains of rhodanese. The shorter segment contains one hydrophobic and one amphiphilic alpha-helix mapping to the domain interface while the other fragment contains the homologous regions and an additional hydrophobic helix. Our results suggest a mechanism for the GroEL-mediated folding of rhodanese in which the domain-forming regions of the polypeptide are kept apart and are then released, perhaps sequentially, resulting in correct folding.

Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH.

The reversible acid denaturation of tumour necrosis factor, TNF alpha, a trimeric, all-beta protein, leads to significant conformational changes within the molecule. A change in far UV CD spectra reveals a shift in the secondary structure content of the protein, with alpha-helical structure being induced. Loss of ellipticity in the near UV reflects a loss of tertiary interactions. This form of TNF is both compact and trimeric, as revealed by fluorescence anisotropy and sedimentation velocity analysis, respectively. Acid-denatured TNF therefore possesses the defining features of the molten globule intermediate while retaining the ability of the still incompletely folded monomers to exhibit the surface specificity necessary for maintaining the trimeric state.

Molecular chaperones in protein folding: the art of avoiding sticky situations.

Molecular chaperones are a class of proteins that interact with the non-native conformations of other proteins. The major role of chaperones of the Hsp70 and Hsp60 families is to prevent aggregation of newly synthesized polypeptides and then to mediate their folding to the native state. As a result of functional studies of these proteins, there has been a revision of the long-held view that protein folding in the cell is a spontaneous process.

Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase. Recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties.

Escherichia coli isocitrate dehydrogenase is completely inactivated by phosphorylation of a single serine residue per subunit. We have examined the conformations of the active and phosphorylated forms of the enzyme using circular dichroism spectroscopy. The results support the view that phosphorylation prevents the binding of NADP, probably by direct blocking of the coenzyme-binding site. Labelling studies suggest that an arginine residue at the coenzyme-binding site may be close to the phosphorylatable serine residue. The phosphorylation of isocitrate dehydrogenase is thus unusual in that it occurs at the active site of the enzyme. We therefore investigated the recognition of isocitrate dehydrogenase by isocitrate dehydrogenase kinase/phosphatase. The kinase activity of this enzyme can phosphorylate intact isocitrate dehydrogenase but not proteolytic fragments derived from it, nor a synthetic peptide corresponding to the sequence round the phosphorylation site.