NMR assignment of domain 3 of the receptor-associated protein (RAP).
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Carbon Isotopes↗
Biomedical subjects
Yibing Wu
Publications and source records attributed to Yibing Wu.
RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi.
The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events.
Alanine↗
Recognition and processing of cisplatin- and oxaliplatin-DNA adducts.
The cytotoxicity of platinum compounds is thought to be determined primarily by their DNA adducts. Cisplatin and oxaliplatin are structurally distinct, but form the same types of adducts at the same sites on DNA. However, the DNA adducts are differentially recognized by a number of cellular proteins. For example, mismatch repair proteins and some damage-recognition proteins bind to cisplatin-GG adducts with higher affinity than to oxaliplatin-GG adducts, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both DNA polymerase beta (pol beta) and DNA polymerase eta (pol eta) catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta may explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins.
Base Pair Mismatch↗
[Study of chemical constituents of the essential oil from Inula britannica L. by GC-MS].
OBJECTIVE: The chemical components of the essential oil from Inula britannica L., which were collected from Yuncheng area of Shanxi province, were analyzed by GC-MS. METHODS: The essential oil was extracted from Inula britannica L. by steam distillation, the components were separated with the capillary chromatographic columns, the amount of the components from the essential oil was determined by normalization method. The components separated were identified by data search system. The chromatographic conditions were as follows: DB-5 (30m x 0.25mm, 0.25 microm) capillary column; High purity helium was used as carrier gas, and the flow rate was 1.0 ml/min; Column temperature: 70 degrees C keeping 2min, from 70 degrees C to 230 degrees C at rising rate 10 degrees C/min and keeping 10 min; Split ratio 15:1; Injector temperature 250 degrees C. RESULTS: 62 peaks were identified representing 68.4% of the total contents. Main component was 1-Benzoxepin-3-ol,2,3,4,5-tetrahydro-(4. 276% ). CONCLUSION: The method is so reliable, stable, and good reproducible that can be applied to the analysis of the essential oil from Inula britannica L.
Asteraceae↗
NMR solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer duplex.
We have determined, at high resolution, the NMR solution structure of an oxaliplatin-GG DNA dodecamer in the AGGC sequence context by 2D NMR studies. Homonuclear assignment strategies resulted in unambiguous assignment of 203 out of 249 protons, which corresponds to assignment of approximately 81% of the protons. Assignments of H5' and H5" protons were tentative due to resonance overlap. The structure of the oxaliplatin duplex was calculated using the program CNS with a simulated annealing protocol. A total of 510 experimental restraints were employed in the structure calculation. Of 20 calculated structures, the 15 with the lowest energy were accepted as a family. The RMSD of the 15 lowest energy structures was 0.68 A, indicating good structural convergence. The theoretical NOESY spectrum obtained by back-calculation from the final average structure showed excellent agreement with the experimental data, indicating that the final structure was in good agreement with the experimental NMR data. Significant conformational differences were observed between the oxaliplatin-GG 12-mer DNA we studied and all previous solution structures of cisplatin-GG DNA duplexes. For example, the oxaliplatin-GG adduct shows much less distortion at the AG base-pair step than the cisplatin-GG adducts. In addition, the oxaliplatin-GG structure also has a narrow minor groove and an overall axis bend of about 31 degrees, both of which are very different from the recent NMR structures for the cisplatin-GG adducts. These structural differences may explain some of the biological differences between oxaliplatin- and cisplatin-GG adducts.
Antineoplastic Agents↗
Protein interactions with platinum-DNA adducts: from structure to function.
Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol beta and pol eta catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.
Base Sequence↗
NMR study of the conformational transition of cytochrome c upon the displacement of Met80 by exogenous ligand: structural and magnetic characterization of azidoferricytochrome c.
As the exogenous ligand-cytochrome c complexes were purported to represent models for the unfolding intermediate of cytochrome c, NMR spectroscopy has been utilized to study the azide adduct of horse heart cytochrome c. The structure of azidoferricytochrome c was modeled by restrained energy minimization using paramagenetic pseudocontact shifts as constraints. The bound azide moiety was found to be tilted approximately 15 degrees from the heme normal. The displacement of Met80 by the exogenous azide molecule causes large structural rearrangement in the distal cavity. Furthermore, the conformation transition associated with the swing out of the loop containing Met80 and the shift of the 50s-helix increases the solvent accessibility of the heme group. To elucidate the heme electronic structure of the complex, the paramagnetic 13C shifts were analyzed in terms of a model based on the pi molecular orbitals of the heme under perturbed D(4) symmetry. It turned out that the His-Fe bonding provides the protein constraint that orients the in-plane anisotropy in the complex. The electronic properties are in accordance with the calculated magnetic susceptibility anisotropy and the structural information.
Animals↗