Biomedical subjects

Yu-Hou Chen

Publications and source records attributed to Yu-Hou Chen.

5 recordsLinked to original sources

PubMed2006

Critical role of tryptophanyl residues in the conformational stability of goose delta-crystallin.

Delta-crystallin is the major structural protein in avian and reptilian eye lenses but its sequence is highly homologous with the urea cycle enzyme, argininosuccinate lyase (ASL). In previous studies the multi-step unfolding process of this protein in the presence of GdmCl was sensitively probed using tryptophan fluorescence. In this study the contribution of single tryptophan residues to the stability of the local environment was monitored by mutation of two highly conservative tryptophan residues in goose delta-crystallin, Trp 74 and Trp 169. These residues behaved differently in terms of fluorescence intensity and maxima emission wavelength, consistent with their structural location in buried or solvent accessible regions. No gross changes in the secondary structure after mutation were observed, as judged by far-UV CD. The side chains of tryptophan residues in the structure of wild-type goose delta-crystallin possess both hydrophobic and hydrogen bonding interactions. Replacement of the side chain with phenylalanine or alanine led to expose of a hydrophobic area and a reduction in thermal stability; W169A particularly has a T(m) value that was 10 degrees C lower than the wild type enzyme. In the presence of GdmCl, a sharp red shift in fluorescence wavelength due to subunit dissociation can be sensitively detected using a single tryptophan, with the region surrounding W74 undergoing the first transition with a [GdmCl](1/2) of 0.45 M. Further measurement of unfolding curves by CD revealed that the W169A mutant was most unstable with a [GdmCl](1/2) of 0.22 M. From sedimentation velocity analysis, the unstable conformation of the W169A mutant affected the assembly of the quaternary structure. Our studies demonstrate the critical role for the tryptophan residues in stabilizing protein conformations and subunit assembly in delta-crystallin.

Animals↗

PubMed2005

The effect of N-terminal truncation on double-dimer assembly of goose delta-crystallin.

Delta-crystallin is a soluble structural protein in avian eye lenses that confers special refractive properties. In the presence of GdmCl (guanidinium chloride), tetrameric delta-crystallin undergoes dissociation via a dimeric state to a monomeric molten globule intermediate state. The latter are denatured at higher GdmCl concentrations in a multi-state manner. In the present study, the X-ray structure of goose delta-crystallin was determined to 2.8 A (1 A=0.1 nm). In this structure the first 25 N-terminal residues interact with a hydrophobic cavity in a neighbouring molecule, stabilizing the quaternary structure of this protein. When these 25 residues were deleted this did not produce any gross structural changes, as judged by CD analysis, but slightly altered tryptophan fluorescence and ANS (8-anilino-1-naphthalenesulphonic acid) spectra. The dimeric form was significantly identified as judged by sedimentation velocity and nondenaturing gradient gel electrophoresis. This mutant had increased sensitivity to temperature denaturation and GdmCl concentrations of 0.3-1.0 M. This protein was destabilized about 3.3 kcal/mol (1 kcal=4.184 kJ) due to N-terminal truncation. After incubation at 37 degrees C N-terminal truncated proteins were prone to aggregation, suggesting the presence of the unstable dimeric conformation. An important role for the N-terminus in dimer assembly of goose delta-crystallin is proposed.

Amino Acid Sequence↗

PubMed2004

Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system.

Human placental alkaline phosphatase was successfully cloned in the yeast system Pichia pastoris. The recombinant enzyme was over-expressed as a secreted protein in the cultured medium. The enzyme was extremely stable, which resulted in a total recovery of the enzyme activity after the purification process. The purified enzyme preparation was apparently homogeneous as examined by the polyacrylamide gel electrophoresis, analytical gel-permeation chromatography, and analytical ultracentrifugation. The final enzyme preparation showed a purification of 803-fold from the culture medium with a specific activity of 578 U/mg of protein. Fluorescence spectroscopic analyses showed multiple unfolding steps in the urea denaturation process of the homodimeric recombinant enzyme. Extensive conformational change of the enzyme in urea was detected by the analytical ultracentrifugation and the size-exclusive chromatography. The quaternary structure of the enzyme is quite stable. No indication of dissociation was observed after extensive tertiary structural changes.

Alcohol Oxidoreductases↗

PubMed2003

Characterization of the interactions between Asp141 and Phe236 in the Mn2+-l-malate binding of pigeon liver malic enzyme.

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141-->Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ ( K (d,Mn)) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K (d,Mn) for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.

Animals↗

PubMed2003

Equilibrium protein folding-unfolding process involving multiple intermediates.

Mathematical models for the protein folding-unfolding process involving multiple intermediates have been derived. Computer fitting of the experimental data to this model generates various thermodynamic parameters for the folding-unfolding process. In this way, the complex folding-unfolding process of the multi-domain proteins can be analysed in a quantitative way. The application of the folding-unfolding model involving seven stages in human placental alkaline phosphatase is described.

Alkaline Phosphatase↗