Biomedical subjects

Sen-Fang Sui

Publications and source records attributed to Sen-Fang Sui.

17 recordsLinked to original sources

PubMed2006

Escherichia coli CorA periplasmic domain functions as a homotetramer to bind substrate.

CorA is a primary Mg2+ transporter in bacteria, which also mediates influx of Ni2+ and Co2+. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning alpha-helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-PPD indicated that E. coli CorA PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full-length CorA and PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.

Cation Transport Proteins↗

PubMed2006

Complexin is able to bind to SNARE core complexes in different assembled states with distinct affinity.

The formation of the functional SNARE complex in vivo is central to the fast neurotransmitter release at the neuronal terminal. Numerous studies revealed that this process involves progressive assembly of an alpha-helical bundle and is dynamically reversible. So far many proteins directly or indirectly take part in this process. Complexin, one of such factors, has revealed rapid association with the SNARE complex, however, whether or not complexin can interact with partially assembled SNARE complex is critical and yet unknown. Here, we present evidence that complexin is able to bind to various mutant versions of the SNARE complex mimicking its quaternary structure at different assembly stages. In addition, the affinity of complexin for the SNARE complex is correlated with the extent to which the SNARE complex is assembled. These results suggest that complexin is able to bind to SNARE complex before its complete formation.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Cholesterol depletion inhibits the degradation of amyloid beta-peptide in rat pheochromocytoma (PC12) cells.

Amyloid beta-peptide (Abeta) is a very important pathogenic factor for Alzheimer's disease (AD). It is a constitutive product of cellular metabolism and occurs in normal biological fluids, such as CSF and plasma. As the steady state levels of all peptides in vivo are a direct consequence of the balance between their anabolism and catabolism, peptide accumulation can arise not only from increased production but also from decreased breakdown. Although increasing evidence indicates that cholesterol is a risk factor for AD development, the exact mechanisms of cholesterol on Abeta metabolism or deposition have remained unclear. Here, we focus our attention on the effect of cholesterol depletion on the degradation of Abeta1-40. Cholesterol depletion of cultured rat pheochromocytoma (PC12) cells was achieved by methyl-beta-cyclodextrin (MbetaCD) extraction. It was found that cholesterol depletion by MbetaCD extraction can suppress Abeta degradation in PC12 cells.

Amyloid beta-Peptides↗

PubMed2005

Three-dimensional architecture of phycobilisomes from Nostoc flagelliforme revealed by single particle electron microscopy.

Phycobilisomes are protein complexes that harvest light and transfer energy to the photo system. Here, the three dimensional structure of intact phycobilisomes from Nostoc flagelliforme is studied by a combination of negative stain electron microscopy and cryo-electron microscopy. Results show that the intact phycobilisomes are composed of a tricylindrical core and six rods. Each allophycocyanin cylinder presents a double-layered structure when viewed from the side and a triangular shape when viewed from the top. These characteristics indicate that allophycocyanin trimers in the intact phycobilisomes are arranged into hexameric oligomers in a parallel manner.

Cryoelectron Microscopy↗

PubMed2005

Specific interaction between Smad1 and CHIP: a surface plasmon resonance study.

The TGF-beta superfamily signaling pathway regulates many important biological processes, including cell growth, differentiation and embryonic pattern formation. Smad1, a member of this signaling pathway that functions downstream of serine/threonine kinase receptors, has ability to interact with carboxyl terminus of Hsc70-interacting protein (CHIP), which is an E3 ubiquitin ligase in other cases. It has been reported that Smurf1, a member of the Hect family E3 ubiquitin ligases, can target Smad1 to 26S proteasome for degradation. In this paper, we studied the interaction of Smad1 and CHIP by combination of surface plasmon resonance and supported monolayer approach. The specific binding of Smad1 to CHIP indicates that the degradation of Smad1 may also be mediated by CHIP, and CHIP may play an essential role in the TGF-beta signaling pathway.

DNA-Binding Proteins↗

PubMed2004

Trichosanthin's interfacial interactions with phospholipids: a monolayer study.

Lipid monolayer at the air/water interface, as half a membrane, was used here to investigate the interaction between trichosanthin (TCS), a ribosome inactivating protein, and phospholipid membrane. First, the protein adsorption experiments showed that the negatively charged DPPG caused obvious enrichment of TCS beneath the monolayer, indicating electrostatic attraction between TCS and the negatively charged phospholipid. Second, when TCS was incorporated into the phospholipid monolayer, it could not be completely squeezed out until the monolayer collapsed. The results were demonstrated to be irrelative with the phospholipid headgroup, suggesting a strong hydrophobic force between TCS and phospholipid hydrocarbon chain was involved in the interaction. Third, the protein/membrane interaction was further studied with fluorescence microscope. The results showed that TCS could penetrate into both the condensed and the fluid phase of the DPPG monolayer under low pH condition and eventually resulted in a homogeneous phospholipid phase. The breakage of ordered packing of phospholipid by TCS may be responsible for this homogenizing effect.

Adsorption↗

PubMed2003

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer.

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.

Amines↗

PubMed2003

Ring-like pore structures of SecA: implication for bacterial protein-conducting channels.

SecA, an essential component of the general protein secretion pathway of bacteria, is present in Escherichia coli as soluble and membrane-integral forms. Here we show by electron microscopy that SecA assumes two characteristic forms in the presence of phospholipid monolayers: dumbbell-shaped elongated structures and ring-like pore structures. The ring-like pore structures with diameters of 8 nm and holes of 2 nm are found only in the presence of anionic phospholipids. These ring-like pore structures with larger 3- to 6-nm holes (without staining) were also observed by atomic force microscopic examination. They do not form in solution or in the presence of uncharged phosphatidylcholine. These ring-like phospholipid-induced pore-structures may form the core of bacterial protein-conducting channels through bacterial membranes.

Adenosine Triphosphatases↗

PubMed2003

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers.

Neuronal exocytosis is mediated by Ca(2+)-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca(2+)-sensing protein proposed to regulate fusion pore dynamics via Ca(2+)-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca(2+)-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca(2+) does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca(2+).C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are approximately 11 nm long and approximately 11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca(2+) first drives C2A-C2B.membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca(2+)-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.

Animals↗

PubMed2003

Change in pH-dependent membrane insertion characteristics of trichosanthin caused by deletion of its last seven C-terminal amino acid residues.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) that can selectively kill some types of cells at low concentration (0.1-1 nM). The pH-dependent membrane insertion ability of TCS makes it possible that the internalized toxin avoids degradation in lysosomes and further undergoes transportation into the cytosol by some still unidentified mechanism. Here, we show that deletion of C-terminal residues affects interactions of modified TCS (C7-TCS) with lipids and reduces its pH-dependent membrane insertion ability. Fluorescence measurements indicate that at low pH C7-TCS undergoes profound conformational changes that causes exposure of a hydrophobic region and leads to oligomerization of the C7-TCS molecules. The results suggest that the membrane insertion of TCS at low pH might be important for translocation of TCS into the cytosol, which is important for exertion of the RIP activity of TCS. Deletion of the last seven C-terminal residues of TCS would reduce both its RIP activity in vitro and cytotoxicity in vivo, with the degree of decrease being more significant for the cytotoxicity in vivo.

Amino Acid Sequence↗

PubMed2003

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor.

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.

Animals↗

PubMed2003

Trichosanthin induces leakage and membrane fusion of liposome.

Trichosanthin (TCS) is a ribosome inactivating protein with multiple pharmacological properties. Here the interaction between TCS and a phospholipid bilayer is investigated to provide evidence for membrane translocation mechanism of TCS. The results show that TCS can destabilize liposomes made by phospholipids with negatively charged head group. The destabilization effect is pH-dependent and happens only under acidic conditions. Membrane fusion is also seen to accompany the destabilizing process. The interaction between a phospholipid bilayer and C7, a mutant of TCS with 7 residues at its C-terminus deleted, has been investigated. Deleting the C-terminus almost completely abolishes the destabilizing effect of TCS on the phospholipid bilayer, which implicates the C-terminus in the interaction between trichosanthin and the membrane.

Amino Acid Sequence↗

PubMed2002

The effects of protein hydrophobic exposure on the slope of the line in the deltapi vs pi(i) plot.

The deltapi vs pi(i) curve from monolayer surface pressure detection is a powerful method to characterize the membrane insertion. The deltapi vs pi(i) curve is fixed by two parameters, the pi(c) and the slope. The intersected point (pi(c)) of deltapi vs pi(i) curve with abscissa is generally used as a quantitative measure of the membrane insertion ability of a protein. In the current work we demonstrate a correlation between the variation in the slope of the deltapi vs pi(i) curve with protein hydrophobic exposure by performing monolayer experiments on three different proteins, human apolipoprotein H, trichosanthin, and mutant trichosanthin. The value of /slope/ increases gradually following the degree of hydrophobic exposure. These findings suggest that the two parameters, the pi(c) and the slope, will complement each other to interpret the lipid/protein interaction involved in membrane insertion.

Cell Membrane↗

PubMed2002

Polymorphism of structural forms of C-reactive protein.

C-reactive protein (CRP), as a classical member of acute phase reactants, plays a variety of physiological functions in vivo and in vitro. Here we show that CRP exists in different structural forms in solution and on phospholipid membranes. At least three types of CRP structures, i.e., the pentameric ring-like form, the small globulin-like form and the fibril-like structure, were observed by combination of size-exclusion chromatography and electron microscopy. The pentameric ring-like CRPs were discovered mostly on ligand containing membrane in a calcium-dependent manner. The globulin-like monomers found on negatively charged membrane in the absence of calcium exhibited structural stability. The fibril-like structures, that have not been reported before, were formed by face-to-face stacking of pentameric CRP in a number from several to hundreds. The freshly purified CRPs form short single-strand fibrils while those CRPs stored for more than several days form long and bundled fibrils. The different structural forms may convert to each other under certain conditions, which gives some hints as to the structural basis of multiple functions of CRP.

Animals↗

PubMed1999

HPLC and MS Studies of Orientation of Melittin on Phospholipid Membrane.

The orientation of melittin binding to liposomes with or without transmembrane potential was investigated. With a combination of high performance liquid chromatography and mass spectrometry, the membrane association state of melittin was detected by analyzing its trypsin-digested products. It was found that the enzyme could access all the proteolytic sites on protein binding to the liposome without membrane potential, while one proteolytic site on the N-terminal of the protein was blocked after it binding to the liposome with negative transmembrane potential. The results showed that melittin changed its orientation in the latter case.

Journal Article↗

PubMed1996

Interaction of Colicin E(1) with Lipid Membranes.

In order to gain an insight into the mechanism of membrane insertion of the pore-forming protein colicin E(1) and the structure of its membrane-bound form, circular dichroism, fluorescence spectroscopy experiments were carried out. The results revealed the law of conformational changes by lipid membrane induction of the colicin E(1) molecule, and suggest that the lipid membranes with negative charges have a strong inducing action on colicin E(1) molecules. With the induction of membrane, the colicin E(1) molecules in different conformational states can all be recovered to the conformation of membrane insertion in native state. The induction intensity of different kinds of lipids on colicin E(1) was in the following order: DMPG>DMPE>DMPC. The binding of colicin E(1) with lipid vesicles was tight and the membrane-bound proteins were resistant to denaturation.

Journal Article↗

PubMed1996

Studies on the Conformation of Apocytochrome c in Different Folding States Inserted into the Membranes.

Correlation between the folding states of the chicken heart apocytochrome c and its membrane insertion ability was studied by the monolayer experiment. The penetration ability of apocytochrome c decreased with its folding. Intrinsic fluorescence emissions of apocytochrome c interacted with soybean phospholipids liposomes suggested that the conformations of apocytochrome c with different folding states in aqueous solution were also different in the membranes. The conformational differences were further characterized by CD spectra of apocytochrome c interacted with DMPC and DMPG liposomes. The results showed that apocytochrome c, which were randomly coiled in aqueous solution, adopted alpha-helical conformations after interaction with DMPG liposome. The apocytochrome c with a folded state in aqueous solution also adopted alpha-helical conformations, but the alpha-helical contents were less than the former. When DMPC liposomes were used, no distinct conformational change was observed.

Journal Article↗