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

Kenneth Hun Mok, Purdue University

Abstract

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$\sp\circ$C whereas trypsinogen was 63$\sp\circ$C 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.

Degree

Ph.D.

Advisors

Light, Purdue University.

Subject Area

Biochemistry|Biophysics|Chemistry

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