Probing the stability and mechanism for folding of the GrpE1-112 tetrameric deletion mutant of the GrpE protein from E. coli

Insight into the stability and folding of oligomeric proteins is essential to the understanding of protein folding, especially since the majority of proteins found in nature are oligomeric. A deletion mutant of the GrpE protein from Escherichia coli, that contains the first 112 residues (GrpE1-112) of 197 total, is an oligomeric protein forming a tetrameric structure. A four-helix bundle structure is formed via the interaction of an α-helix (22 amino acids in length) from each monomer. Using both thermal and chemical (urea) denaturation studies, the GrpE1-112 protein has rather low stability with a Tm of unfolding of 37 °C, a Cm (urea) of 1.3 M, and a ΔGunfolding of 8.4 kJ mol−1. Investigation into the folding pathway using circular dichroism (CD) stopped-flow revealed a two step process with a fast first phase (krefolding = 8.0 × 106 s−1 M−1) forming a multimeric intermediate that possesses significant α-helical content followed by a slow, first order, step forming the folded tetramer.

► The stability and folding of a tetrameric deletion mutant of GrpE was studied by CD and CD stopped-flow.
► The GrpE1-112 mutant has low stability with a midpoint of unfolding of 37 °C.
► Equilibrium studies on GrpE1-112 did not reveal any stable dimeric intermediate upon protein unfolding.
► The folding pathway involves a two step process with a fast first phase forming a multimeric intermediate.
► The intermediate contains the majority of overall α-helical structure and is followed by a second slow phase to form the tetramer species.