Hyeong Ju Lee

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase.

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two‐state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two‐state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X‐ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7 Å in 0 M urea, 17.3 Å in 5.2 M urea, and 25.1 Å in 7 M urea by SAXS. The size of KSI in 5.2 M urea was significantly decreased compared with those in 0 M and 7 M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2 M urea is distinctly different from native and fully‐unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using 1H NMR spectroscopy and Kratky plot supported the existence of a partially‐folded form of KSI which is distinct from those of native and fully‐unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.

15N NMR Relaxation Studies of Y14F Mutant of Ketosteroid Isomerase : The Influence of Mutation on Backbone Mobility

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

Thermodynamics of partitioning of substance P in isotropic bicelles

The temperature dependence of the partition of a neuropeptide, substance P (SP), in isotropic (q = 0.5) bicelles was investigated by using pulsed field gradient NMR diffusion technique. The partition coefficient decreases as the temperature is increased from 295 to 325 K, indicating a favorable (negative) enthalpy change upon partitioning of the peptide. Thermodynamic analysis of the data shows that the partitioning of SP at 300 K is driven by the enthalpic term (ΔH) with the value of − 4.03 kcal mol−1, while it is opposed by the entropic term (−TΔS) by approximately 1.28 kcal mol−1 with a small negative change in heat capacity (ΔCp). The enthalpy‐driven process for the partition of SP in bicelles is the same as in dodecylphosphocholine (DPC) micelles, however, the negative entropy change in bicelles of flat bilayer surface is in sharp contrast with the positive entropy change in DPC micelles of highly curved surface, indicating that the curvature of the membrane surface might play a significant role in the partitioning of peptides. Copyright

Contribution of a low-barrier hydrogen bond to catalysis is not significant in ketosteroid isomerase.

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3-ketosteroid to its conjugated Δ4-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.

NMR Studies on the Equilibrium Unfolding of Ketosteroid Isomerase by Urea

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.