Further characterization of refolding intermediates of bovine trypsinogen: Identification of thermodynamically unstable globular intermediates

A physico-chemical characterization of the refolding intermediates of bovine trypsinogen is presented. Size-exclusion HPLC was used to partially separate refolding intermediates. Spectral and stability studies performed on these intermediate fractions have shown the following. (1) Far-UV circular dichroism showed that the spectra of the intermediates were similar with one another while considerably different from the unfolded Tg(SSG)\sb{12} and completely folded native molecules. It is suggested that $\beta$-sheet structure was present, although this secondary structure may be different from the native $\beta$-sheets found in trypsinogen. (2) UV and fluorescence spectra confirm that the intermediates are clearly distinguishable from Tg(SSG)\sb{12} and trypsinogen, while exhibiting similar burial of aromatic side chains. (3) Approximately five of the six disulfide bonds stabilized the intermediates, however these disulfide bonds were non-native. (4) Enzymatic activity was present only in the completely folded native molecule fractions suggesting that there were no near-native conformations. (5) The hydrophobic fluorescence probe, 1-anilinonaphthalene-8-sulfonate (ANS), bound strongly to these intermediates, indicating that they had exposed hydrophobic regions. (6) A method monitoring the release of bound ANS upon thermal denaturation of the intermediates was developed to measure the stabilities of these molecules. The T\sb{\rm m} values of the intermediates were approximately 20\sp\circC whereas trypsinogen was 63\sp\circC under the same conditions. These molecules were extremely unstable and therefore capable of interconverting to different conformations. A folding pathway is proposed. Unfolded trypsinogen-glutathione mixed disulfide rapidly becomes compact, and the partially-folded intermediates sample conformational space via disulfide bond interchange. The stabilities of these molecules are low ($\Delta$G\sp\circ\sim 0.4 kcal/mol) so multiple structures could be randomly tested until native-like tertiary interactions are satisfied. When this occurs, the negative $\Delta$G\sp\circ will drive the intermediates to fold into the stable, native structure. Thermal stabilities of the homologous pancreatic serine proteases trypsinogen, chymotrypsinogen A, and elastase were measured and calculated $\Delta$G\sp\circ values were compared. It is argued that the stabilities of the three proteins differ primarily because of the number of disulfide bonds (six, five, and four, respectively) present in each molecule. However, disulfide bond formation does not seem to be the rate-limiting step in acquiring native structure from the unfolded state. Rather, correct native-like tertiary interactions appear to be responsible for the slowest step in folding

Structure and function of human α-lactalbumin made lethal to tumor cells (HAMLET)-type complexes.

Human α-lactalbumin made lethal to tumor cells (HAMLET) and equine lysozyme with oleic acid (ELOA) are complexes consisting of protein and fatty acid that exhibit cytotoxic activities, drastically differing from the activity of their respective proteinaceous compounds. Since the discovery of HAMLET in the 1990s, a wealth of information has been accumulated, illuminating the structural, functional and therapeutic properties of protein complexes with oleic acid, which is summarized in this review. In vitro, both HAMLET and ELOA are produced by using ion-exchange columns preconditioned with oleic acid. However, the complex of human α-lactalbumin with oleic acid with the antitumor activity of HAMLET was found to be naturally present in the acidic fraction of human milk, where it was discovered by serendipity. Structural studies have shown that α-lactalbumin in HAMLET and lysozyme in ELOA are partially unfolded, 'molten-globule'-like, thereby rendering the complexes dynamic and in conformational exchange. HAMLET exists in the monomeric form, whereas ELOA mostly exists as oligomers and the fatty acid stoichiometry varies, with HAMLET holding an average of approximately five oleic acid molecules, whereas ELOA contains a considerably larger number (11- 48). Potent tumoricidal activity is found in both HAMLET and ELOA, and HAMLET has also shown strong potential as an antitumor drug in different in vivo animal models and clinical studies. The gain of new, beneficial function upon partial protein unfolding and fatty acid binding is a remarkable phenomenon, and may reflect a significant generic route of functional diversification of proteins via varying their conformational states and associated ligands