An unfolding/refolding step helps in the crystallization of a poorly soluble protein.
Proteins that are unstable or poorly soluble often elude crystallization. Here, a novel strategy is presented that leads to the crystallization of the isolated N-terminal propeptide of human procathepsin S, a proteinase belonging to the cathepsin L-like endopeptidases of the clan CA1 cysteine peptidases. Being very hydrophobic, the propeptide is extremely poorly soluble in aqueous solvents at neutral pH. Solubility is much better at acidic pH, but the native structure is destroyed under these conditions. A novel approach to the crystallization of this poorly soluble protein is presented in which it is first unfolded in an acidic buffer (pH 4.5) and then mixed with a nearly neutral crystallization buffer (pH 6.75) in which the native conformation should form spontaneously. Crystals were grown at a high concentration of MES (1.14 M) with 10% 2-propanol as precipitant. They belong to a tetragonal space group, with unit-cell parameters a = b = 151.1, c = 75.8 A. Diffraction data to a resolution of 3.5 A were obtained.