Yong Je Chung

Conformational Preferences of X-Pro Sequences: Ala-Pro and Aib-Pro Motifs

Conformational preferences and prolyl cis-trans isomerizations of the X-Pro motifs (Ac-X-Pro-NHMe, X = Ala and Aib) are explored using the meta-hybrid functional M06-2X and the double-hybrid functional B2PLYP-D with empirical dispersion corrections in the gas phase and in water, where solvation free energies were calculated using the implicit SMD model. Ac-Ala-Pro-NHMe favors the type VI β-turns in the gas phase and the open conformations in water. The populations of type VI β-turns decrease from 71% in the gas phase to 21% in water, which is reasonably consistent with IR and NMR experimental results on tBoc-Ala-Pro-NHMe. However, Ac-Aib-Pro-NHMe prefers the type I β-turns with α-helical structures for both residues in the gas phase and in water, whose populations are estimated to be 66% in both phases. These calculated results may rationalize why most of the peptaibiotics containing the Aib-Pro sequence have a regular α-helical conformation at the N- or C-terminus but a kinked α-helical structure in the middle of the helix. The cis-trans isomerizations of the Ala-Pro and Aib-Pro peptide bonds proceed via the clockwise rotation with the different backbone conformations. The rotational barriers to cis-to-trans isomerization are estimated to be 19.73 kcal/mol for the Ala-Pro tripeptide and 16.64 kcal/mol for the Aib-Pro tripeptide in water, which indicates that the rotational barrier becomes lower by ~3 kcal/mol for the Aib-Pro peptide bond. The calculated rotational barrier for Ac-Ala-Pro-NHMe is consistent with the observed value of 19.3 kcal/mol for Suc-Ala-Ala-Pro-Phe-pNA from NMR experiments in a buffered solution.

Molecular cloning and characterization of WD40-repeat protein from Clonorchis sinensis

WD40-repeat proteins have four to eight repeating units flanked by Gly-His (GH) and Trp-Asp (WD) at both termini and folds into a β-propeller. A polypeptide deduced from a Clonorchis sinensis cDNA clone analyzed to have seven WD40-repeats and predicted to form a β-propeller (CsWD1). The CsWD1 protein was expressed stage-specifically in the metacercariae and localized in the tegumental syncytium. The CsWD1 protein is suggested to serve as a platform for interacting partner proteins in the tegumental syncytium of C. sinensis metacercariae.

Crystallization of Clonorchis sinensis 26 kDa glutathione S‐transferase and its fusion proteins with peptides of different lengths

A Clonorchis sinensis 26 kDa glutathione S-transferase (CsGST) and its fusion proteins containing 14 and 48 amino-acid peptides at the N-terminus have been crystallized using polyethylene glycol monomethylether 550 as a precipitant. Crystals of the three proteins show very similar crystal properties: they diffract to at least 2.3 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters of CsGST crystals were a = 66.64 (1), b = 68.91 (1), c = 123.41 (2) A, which are very close to those of the crystals of the two fusion proteins. In addition, CsGST fusion proteins containing varying extents of N-terminal-extended peptides are incorporated into a crystal, indicating that the extended peptides have little effect on crystal packing. These results suggest that the crystallization system of CsGST/peptide fusion protein may be generally applicable to obtain crystals of small peptides.

Crystal structures of 26kDa Clonorchis sinensis glutathione S-transferase reveal zinc binding and putative metal binding.

The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST-D26H structure showed that four histidine residues - His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule - coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.

A histidine substitution confers metal binding affinity to a Schistosoma japonicum Glutathione S-transferase

Glutathione S-transferases (GSTs) are multifunctional enzymes that are used as fusion tags on recombinant proteins in mammalian and Escherichia coli expression systems. We recently found that the Schistosoma japonicum GST (SjGST) displays weak Ni(2+) ion binding affinity. Glu26 and His79 were assumed to be its Ni(2+) binding sites based on the structure of the 26-kDa Clonorchis sinensis GST. To enhance SjGST Ni(2+) binding affinity, Glu26 was mutated to His. SjGST-E26H was expressed and purified at a high concentration of imidazole to a higher purity than wild type SjGST. In addition, human biotin protein ligase fused to SjGST-E26H was purified with a immobilized Ni affinity column.

Investigating the binding area of protein surface using MCL algorithm

Proteins combine with other materials to achieve a variety of functions, which will be similar if their active sites are similar. Thus we can infer a proteinpsilas function by identifying its binding area. This paper proposes a novel method to select a proteinpsilas binding area using the Markov Cluster (MCL) algorithm. A distance matrix is constructed from the surface residues distance on the protein, then transformed to the connectivity matrix for application of the MCL process, and finally evaluated by using Catalytic Site Atlas (CSA) data. In the experimental result using CSA data which comprised 94 selected single chain proteins, our algorithm detects 91 (97%) binding areas near the active site of each protein. We introduced new geometrical features with the aim of improving the prediction accuracy of the active site residues by selecting the residues near the active site.