Biomedical subjects

Hong-Yu Hu

Publications and source records attributed to Hong-Yu Hu.

24 records · Page 2Linked to original sources

PubMed2003

Crystallization and preliminary crystallographic studies of Escherichia coli CcmG/DsbE protein.

CcmG/DsbE is a typical thiol/disulfide oxidoreductase, exhibiting a specific reducing activity in a highly oxidizing environment, and is involved in electron transfer during the maturation of c-type cytochromes. Escherichia coli CcmG/DsbE (residues 19-185) has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.48, b = 48.52, c = 84.78 A. X-ray data have been collected to 1.9 A resolution.

Crystallization↗

PubMed2003

A peptide motif consisting of glycine, alanine, and valine is required for the fibrillization and cytotoxicity of human alpha-synuclein.

Amyloid-like aggregation or fibrillization of alpha-synuclein (alpha-Syn) and the filamentous deposits in Lewy bodies are believed to be closely associated with several fatal neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Here, we report the importance of a nine-residue peptide motif, (66)VGGAVVTGV(74), in the fibrillization and cytotoxicity of human alpha-Syn. Mutagenesis combined with thioflavin T fluorescence detection, atomic force microscopic imaging, and cytotoxicity assays reveal that deletion of this sequence completely eliminates alpha-Syn fibrillization and cell toxicity. However, deletion of the (71)VTGV(74) sequence decreases the fibrillization rate while the cytotoxicity remains unchanged. Incorporation of charged residues within this region slows aggregation and even impedes filament formation. In addition, substitution of Gly68 with Ala or C-terminal truncations of alpha-Syn accelerate the fibrillization processes. Circular dichroism studies suggest that beta-sheet formation is often concomitant with filament formation. Thus, this segment, namely, the GAV motif, is responsible for aggregation or fibrillization of alpha-Syn and perhaps other amyloidogenic proteins. The oligomers formed during fibrillogenesis might be associated with the cytotoxicities of various alpha-Syn species. This finding may provide further insight into the understanding of the molecular mechanism underlying the fibrillogenesis implicated in neurodegeneration as well as aid in drug design and development of transgenic models.

Alanine↗

PubMed2003

Study of protein-protein interactions by fluorescence of tryptophan analogs: application to immunoglobulin G binding domain of streptococcal protein G.

The protein-protein interaction system often contains many fluorophores that may significantly interfere with the quantitative determination of the binding abilities. To solve this perplexing problem, we biosynthetically incorporated the two tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into the immunoglobulin G (IgG) binding domain of streptococcal protein G (PGBD). The exclusive excitation and novel fluorescence changes in both the intensity and anisotropy are beneficial to reporting the details of the interactions between PGBD and the IgG fragments and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 microM for the binding of human Fc and 8.0 microM for mouse Fc. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the binding abilities and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex.

Bacterial Proteins↗

PubMed2003

[NMR study on solution configuration of mHCN1 pore peptide].

Spin systems for amino acid residues in mHCN1 pore region peptide have been identified through analysis of 2D NMR spectra. The sequence-specific assignment of spin systems was obtained by NOEs correlation in WET-NOESY spectra, and the complete assignment of proton resonances for backbone and side chain has been achieved. CNS software was used to calculate the structure of mHCN1 19 aa peptide. The results show that an alpha-helix from residue 10 to residue 13 is formed within the pore region. The results of NMR study on mHCN1 pore peptide provide the basis for further understanding the mechanism of ion selectivity of channels.

Amino Acid Sequence↗

PubMed2002

Structural transformation and aggregation of human alpha-synuclein in trifluoroethanol: non-amyloid component sequence is essential and beta-sheet formation is prerequisite to aggregation.

Amyloid-like aggregation of alpha-synuclein and deposit in Lewy bodies are thought to be the major cause of Parkinson's disease. Here we describe the secondary structural transformation and aggregation of human alpha-synuclein and its C-terminus truncated fragments in trifluoroethanol. Proteins containing the NAC (non-amyloid component) segment undergo a three-state transition: from native random coil to beta-sheet and to alpha-helical structure, while the NAC deficient fragment and gamma-synuclein undergo a typical two-state coil-to-alpha transition. The beta-sheet form is highly hydrophobic that strongly binds to 1-anilinonaphthalene-8-sulfonic acid (ANS) and is prone to self-aggregation. The results suggest that the NAC sequence is essential to beta-sheet formation and the aggregation originates from the beta-sheet intermediate, which may be implicated in the pathogenesis of Parkinson's disease.

Biopolymers↗

PubMed2002

[Redox properties and conformational changes of DsbA protein from Escherichia coli periplasm].

DsbA protein, a disulfide bond enzyme in Escherichia coli periplasm, catalyzes mainly disulfide bond formation of proteins. Site-directed mutagenes is combined with Trp-analog labeling technique was used to investigate the redox properties and conformational changes of DsbA. The results show that: (1) DsbA, as an oxidase, can catalyze disulfide bond formation efficiently. The reduced form is more stable than the oxidized form, suggesting that the strong oxidizing force of DsbA comes from the tense conformation of the oxidized form. (2) The alteration of local environment around Trp(76) between redox forms is responsible for the special fluorescence phenomenon of DsbA. (3) The result from (19)F-NMR study provides further evidence that the local conformation of Trp(76) is dramatically affected during the transformation of redox forms, but that of Trp(126) remains unaffected.

Circular Dichroism↗