Most protease prosegments are co-synthesized at the N-termini of cysteine proteases and are involved in folding assistance, inhibition, and activation of their mature enzymes. By using circular dichroism, UV-difference and fluorescence spectroscopies, we studied the thermal unfolding of papain prosegment. The transition seems to be two-state and reversible, with an unfolded state prone to aggregation. Unfolding thermodynamic parameters obtained show low values both for Delta H-Tm and Delta C-pU, indicative of a loosely packed three-dimensional conformation for the prosegment at near-neutral pH conditions. In spite of these results, fluorescence experiments demonstrate that papain prosegment is able to recognize and inhibit its cognate protease. An acid medium induces a molten globule-like state without intermediates, which in turn undergoes an irreversible thermal unfolding. Our results suggest that papain prosegment has a high degree of conformational flexibility, with the ability to form not only a molten globule-like structure in activating conditions, but also requiring an induced fit in order to be functional as inhibitor. (c) 2005 Elsevier Inc. All rights reserved.
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