INVESTIGATION OF THE FOLDING PATHWAY OF THE TEM-1 BETA-LACTAMASE

The TEM-1 beta-lactamase is a globular protein containing 12 proline r esidues, The folding mechanism of this enzyme was investigated by kine tic and equilibrium experiments with the help of fluorescence spectros copy and circular dichroism. The equilibrium denaturation of the prote in induced by guanidine hydrochloride occurs in two discrete steps, in dicating the existence of a thermodynamically stable intermediate stat e. This state is 5.2 +/- 0.4 kcal/mol less stable than the native conf ormation and 5.7 +/- 0.2 kcal/mol more stable than the fully denatured protein, This intermediate state exhibits a high content of native se condary structure elements but is devoid of specific tertiary organiza tion; its relation to the ''molten globule'' is discussed. Refolding k inetic experiments revealed the existence of a transient intermediate conformation between the thermodynamically stable intermediate and the native protein, This transient intermediate appears rapidly during th e folding reaction. It exhibits a secondary structure content very sim ilar to that of the native protein and has also recovered a significan t amount of tertiary organisation. The final refolding step of the TEM -1 beta-lactamase, leading to the native enzyme, is dominated by two m ajor slow kinetic phases which probably reflect a very complex process kinetically Limited by proline cis/trans isomerization. (C) 1995 Wile y-Liss, Inc.