NMR studies on the structural characteristics of unfolding of Δ5-3-ketosteroid isomerase

Abstract

Proteins can be denatured or misfolded by some factors such as heat, pH and denaturing agents, which cause not only the disappearance or change of their functions but also a number of fatal diseases. In order to diagnose and cure the diseases, it is necessary to understand the structural characteristics of protein folding or unfolding. The subject of this thesis is to elucidate the structural characteristics of Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida biotype B at initial stage of unfolding using NMR spectroscopy. In order to investigate the detailed structural characteristics of KSI during unfolding, the active site mapping has been done by analysis of paramagnetic effect in 1H-15N HSQC spectra using hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediate analog (equilenin). The results revealed that residues in hydrophobic cavity of KSI, particularly active site region, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while they experienced full recovery of a lineshape upon the addition of equilenin. The mapped region resembles to the active site of KSI as described by the crystal structure. These observations indicate that a combined use of paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site of KSI. NMR studies on the structural characteristics of KSI unfolding were also performed using urea, HyTEMPO and equilenin. The results show that HyTEMPO may selectively induce line-broadening of NMR signals of some residues at initial stage of KSI unfolding by urea. However, the use of paramagnetic molecule for unfolding study of the protein is somewhat limited, since urea could also lead to line-broadening due to the chemical exchange between two different sites. The effect of denaturing factors such as guanidinium hydrochloride (GdmHCl) and temperature has also been studied focusing on the initiation site of KSI unfolding. In particular, subtle structural changes in early stage of KSI unfolding were examined using 1H-15N HSQC and circular dichroism (CD) measurements. Analysis of the chemical shift changes at various conditions of denaturing factors revealed that the residues in hydrophobic cavity of KSI were more significantly perturbed by GdmHCl or temperature than other regions. The results suggest two features

namely, β - 1 and β - 3 strands were perturbed first by GdmHCl in KSI, while with temperature, it was a combination of both all α - helices and β - 3 and β - 6 strands. Moreover, though the effect of denaturing factors is quite different, residues which formed the entrance of the hydrophobic cavity of KSI were initially destabilized by both GdmHCl and temperature, suggesting the entrance region as an initiation site of KSI unfolding. These results could help provide detailed insight into the initiation site of unfolding of dimeric protein at residue level.