Biomedical subjects

Hui-Ting Chou

Publications and source records attributed to Hui-Ting Chou.

4 recordsLinked to original sources

PubMed2006

Milestones in electron crystallography.

Electron crystallography determines the structure of membrane embedded proteins in the two-dimensionally crystallized state by cryo-transmission electron microscopy imaging and computer structure reconstruction. Milestones on the path to the structure are high-level expression, purification of functional protein, reconstitution into two-dimensional lipid membrane crystals, high-resolution imaging, and structure determination by computer image processing. Here we review the current state of these methods. We also created an Internet information exchange platform for electron crystallography, where guidelines for imaging and data processing method are maintained. The server (http://2dx.org) provides the electron crystallography community with a central information exchange platform, which is structured in blog and Wiki form, allowing visitors to add comments or discussions. It currently offers a detailed step-by-step introduction to image processing with the MRC software program. The server is also a repository for the 2dx software package, a user-friendly image processing system for 2D membrane protein crystals.

Cryoelectron Microscopy↗

PubMed2006

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli.

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.

Adenosine Triphosphatases↗

PubMed2006

Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex.

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2002

Solution structure and dynamics of the lipoic acid-bearing domain of human mitochondrial branched-chain alpha-keto acid dehydrogenase complex.

The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain alpha-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex. We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain alpha-keto acid dehydrogenase complex (hbLBD). Similar to LBD from other members of the alpha-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened beta-barrel formed by two four-stranded antiparallel beta-sheets. The lipoyl Lys(44) residue resides at the tip of a beta-hairpin comprising a sharp type I beta-turn and the two connecting beta-strands 4 and 5. A prominent V-shaped groove formed by a surface loop, L1, connecting beta 1- and beta 2-strands and the lipoyl lysine beta-hairpin constitutes the functional pocket. We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from (15)N-T(1), (15)N-T(2), and ((1)H-(15)N) nuclear Overhauser effect data obtained at 600 MHz. The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys(44) beta-turn are highly flexible, whereas beta-sheet S1 appears to display a slow conformational exchange process.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗