PubMed Health⌕ Search

Biomedical subjects

Zhong-Xian Huang

Publications and source records attributed to Zhong-Xian Huang.

16 recordsLinked to original sources
PubMed2006

Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3.

Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, until now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is the first report that other residues, besides the TCPCP motif, in the beta-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [1H-15N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.

Animals↗
PubMed2006

The role of Thr5 in human neuron growth inhibitory factor.

GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer's disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.

Animals↗
PubMed2006

Solution structure and dynamics of human metallothionein-3 (MT-3).

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.

Alzheimer Disease↗
PubMed2005

Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b5).

The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b5 (cyt b5) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b5) using the constructed expression vector pGEX-cyt b). The GST-cyt b5 fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b5 during the fermentation, and the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. The fusion protein was characterized by its spectroscopic and electrochemical properties, the interaction between GST-cyt b5 and cyt c was also investigated. The results show that GST-cyt b5 fusion protein shares similar properties and functions to that of isolated cyt b5. Although cyt b5 and GST were fused together, the two partners have not made significant structural and functional alterations of their counterparts, the protein-protein interactions between them are apparently very weak. To our knowledge, the present study is the first report to express cyt b5 as a GST-cyt b5 fusion protein, which provides a good example for the in vivo maturation of a hemoprotein as a GST fusion protein and sheds new light on the protein-protein interactions within the GST fusion protein.

Amino Acid Sequence↗
PubMed2005

The properties of the metal-thiolate clusters in recombinant mouse metallothionein-4.

Metallothioneins (MTs) are metal-binding proteins with low molecular weight and conservative cysteine residues. Metallothionein-4 (MT-4), one of MT isoforms, is first reported to be distributed in a tissue-specific manner, mainly in stratified squamous epithelia. Here, we compare the properties of metal-thiolate clusters in MT-4 to those in MT-1 and MT-3, including the stabilities toward both pH change and EDTA, as well as the exposure of thiolates to solvent. The metal-thiolate clusters in MT-3 show different property and activity to the reactions compared with MT-4 and MT-1. The structure of metal-thiolate clusters in MT-4 is similar to that of MT-1 from the UV and CD spectra. During pH titration and DTNB reaction, MT-4 and MT-1 exhibit comparable behavior. But while reacting with EDTA, the metal-thiolate clusters in MT-4 are more stable than those of MT-1. We suppose the negative charge of the beta-domain of MT-4 prevents the EDTA attack to MT-4.

Animals↗
PubMed2004

Mapping the electron transfer interface between cytochrome b5 and cytochrome c.

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.

Amino Acid Substitution↗
PubMed2004

The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H.

A comparative study on the solution structures of bovine microsomal cytochrome b5 (Tb5) and the mutant V45H has been achieved by 1D and 2D 1H-NMR spectroscopy to clarify the differences in the solution conformations between these two proteins. The results reveal that the global folding of the V45H mutant in solution is unchanged, but the subtle changes exist in the orientation of the axial ligand His39, and heme vinyl groups. The side chain of His45 in V45H mutant extends to the outer edge of the heme pocket leaving a cavity at the site originally occupied by the inner methyl group of Val45 residue. In addition, the imidazole ring of axial ligand His39 rotates counterclockwise by approximately 3 degrees around the His-Fe-His axis, and the 4-heme vinyl group turns to the space vacated by the removed side chain due to the mutation. Furthermore, the helix III of the heme pocket undergoes outward displacement, while the linkage between helix II and III is shifted leftward. These observations are not only consistent with the pattern of the pseudocontact shifts of the heme protons, but also well account for the lower stability of V45H mutant against heat and urea.

Amino Acid Substitution↗
PubMed2003

The solution structure of the oxidized bovine microsomal cytochrome b(5) mutant V61H.

Using 1488 NOE constraints, 19 stereo-specific assignments, 13 pairs of H-bond constraints, and 140 pseudo-contact shift constraints, a family of 35 structures of bovine microsomal cytochrome b(5) mutant V61H has been obtained through the program PSEUDYANA. The family has been further refined by restrained energy minimization to give a family of final structures. The RMSD values of final structures with respect to the average structure are 0.45+/-0.11 and 0.96+/-0.10A for backbone and heavy atoms, respectively. The final Deltachi(ax) and Deltachi(rh) values are 2.34 x 10(-32) and -0.67 x 10(-32)m(3), respectively. The comparisons between the solution structures of mutant V61H and WT cytochrome b(5), and X-ray structure of the mutant V61H show that the global folding of the molecule in solution is unchanged and the side-chain of His61 deviates from the heme pocket and extends into the solvent like in its crystal structure. However, the helices around the heme pocket undergo outward global displacement while their local conformations are well maintained. Meanwhile, the heme ring shows a little off the heme pocket, which accounts for the lower stability of the mutant. Additionally, the axial ligand rings counterclockwise rotate around His39 N-Fe axis due to the mutation, which is confirmed by variation of the hyperfine shifts of the heme protons of V61H compared to those of WT cytochrome b(5).

Amino Acid Sequence↗
PubMed2003

1H NMR studies of the effect of mutation at Valine45 on heme microenvironment of cytochrome b5.

1D and 2D (1)H NMR were employed to probe the effects on the heme microenvironment of cytochrome b(5) caused by the mutation from Val45 to Tyr45, His45 and Glu45. Compared with wild type (WT) cytochrome b(5), in all mutants the heme ring are CCW rotated relative to the imidazole planes of axial ligands and the angles beta between two axial ligand imidazole planes are not changed, being in agreement with the temperature dependence of the shifts of the heme protons. The ratios of heme isomers (major to minor) are smaller than that in WT. The 4-vinyl group of the heme in V45Y assumes cis-orientation, being similar to that of WT, while in V45E and V45H, both cis and trans orientation are found. The relationships between the structure and biological function of the mutants are discussed in terms of the geometry of heme and axial ligands, the hydrophobicity of heme pocket and the electrostatic potential of the heme-exposed area.

Animals↗
PubMed2003

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies.

To investigate the contribution of tryptophan-121 (Trp121) residue to the structure and function of soluble CuA domain of cytochrome c oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deleted mutant of the soluble domain of Paracoccus versutus cytochrome c oxidase, were constructed and expressed in Escherichia coli BL21 (DE3). Optical spectral studies showed that both the coordination structure of the CuA center and the secondary structure of the protein were changed significantly in the Leu substitution and deletion mutants of Trp121. Their electron transfer activity with cytochrome c was inhibited severely, as shown in stopped-flow kinetic studies. However, the CuA center can be reconstructed in the Trp121Tyr mutant although its stability decreases compared with the wild-type protein. This mutant keeps the same secondary structure as the wild-type protein, but can only transfer electrons with cytochrome c at a rate of one-seventh-fold. Based on the information on the structure, we also investigated and analyzed the possible factors that affect electron transfer. It appears that the aromatic ring, the size of the side chain and the hydrogen bonding ability of the Trp121 are crucial to the structure and function of the soluble CuA domain.

Animals↗
PubMed2003

The effect of the EAAEAE insert on the property of human metallothionein-3.

MT3 shows apparently different properties and function from MT1 even though they have 70% sequence homology. Possibly the two inserts, Thr5 and a negatively charged hexapeptide at position-55 in MT3, play important roles. A series of MT3 variants around the EAAEAE hexapeptide have been prepared by site-directed mutagenesis and their properties and reactivity towards pH, EDTA and DTNB have been studied. Our detailed studies revealed that the EAAEAE insert is essential to the property of MT3. It is the hexapeptide insert, to some extent, making the MT3 alpha-domain looser and lower stability of the metal-thiolate cluster, which could be accessed more easily.

Dithionitrobenzoic Acid↗
PubMed2003

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5.

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) >> His39Cys > His39Ser.

Animals↗
PubMed2002

Expression, characterization, and reaction of recombinant monkey metallothionein-1 and its C33M mutant.

After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system. The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant). The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra. The reactions of MI-1 and C33M mutant with 5,5'-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied. The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra. The mutation of cysteine-to-methionine at position 33 mostly maintains the alpha-domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain.

Amino Acid Sequence↗
PubMed2002

X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35-->Tyr mutant.

Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b5 (cyt b5). This patch is partially exposed on the surface of cyt b5 while its buried face is in direct van der Waals' contact with heme b. Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site-directed mutagenesis we have produced three mutants of cyt b5: Phe35-->Tyr, Phe35-->Leu, and Phe35-->His. We found that of these three mutants, the Phe35-->Tyr mutant displays abnormal properties. The redox potential of the Phe35-->Tyr mutant is 66 mV more negative than that of the wild-type cyt b5 and the oxidized Phe35-->Tyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b5. In this study we studied the most interesting mutant, Phe35-->Tyr, by X-ray crystallography, thermal denaturation, CD and kinetic studies of heme dissociation to explore the origin of its unusual behaviors. Analysis of crystal structure of the Phe35-->Tyr mutant shows that the overall structure of the mutant is basically the same as that of the wild-type protein. However, the introduction of a hydroxyl group in the heme pocket, and the increased van der Waals' and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35-->Tyr mutant. The kinetic difference of the heme trapped by the heme pocket also supports this conclusion. The detailed conformational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence of the folding intermediate.

Animals↗
PubMed2001

Kinetic studies on the oxidation of cytochrome b(5) Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes.

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.

Cytochrome c Group↗