Existence Of Conformational Differences Research Articles

Solid-Phase Total Synthesis of Cherimolacyclopeptide E and Discovery of More Potent Analogues by Alanine Screening

Cherimolacyclopeptide E (1) is a cyclic hexapeptide obtained from Annona cherimola, reported to be cytotoxic against the KB (human nasopharyngeal carcinoma) cell line. The solid-phase total syntheses of this cyclic peptide and its analogues were accomplished by employing FMOC/tert-butyl-protected amino acids and the Kenner sulfonamide safety-catch linker. The synthetic peptide 1 was found to be weakly cytotoxic against four cell lines (MOLT-4, Jurkat T lymphoma, MDA-MB-231, and KB). Analogues 3 and 7, where glycine at positions 2 and 6 of the parent compound was replaced by Ala, exhibited enhanced cytotoxicity against KB (3, IC50 6.3 μM; 7, IC50 7.8 μM) and MDA-MB-231 breast cancer cells (3, IC50 10.2 μM; 7, IC50 7.7 μM), thereby suggesting possible selective targeting of these cancer cells by these peptides. The spectral data of synthetic peptide 1 was found to be similar to that reported for the natural product. However, a striking difference in biological activity was noted, which warrants the re-evaluation of the original natural product for purity and the existence of conformational differences.

Short Communication: Identification of the Conformational Requirement for the Specificities of Coreceptors for Human and Simian Immunodeficiency Viruses

More than 10 G protein-coupled receptors (GPCRs) work as coreceptors for human and simian immunodeficiency viruses (HIVs/SIVs); however, structural features critical for coreceptor activity have not been identified. Our objective was to elucidate the structural requirement of coreceptor activities. Amino-terminal regions (NTRs), extracellular loops (ECLs), and the undecapeptidyl arch (UPA) in the second ECL have been shown to be important for coreceptor function. We made chimeric coreceptors for these regions between CCR5 and GPR1, which is genetically distant from CCR5, and analyzed their activities. The coreceptor activity and specificity of CCR5 were maintained when its NTR or UPA was replaced with GPR1. In contrast, the GPR1 chimera with CCR5 NTR was used by HIV-1 strains that can use only CCR5, but not both CCR5 and CXCR4, or GPR1. GPR1 chimera with CCR5 UPA almost lost activity. All ECL chimeras could hardly maintain activity. Thus, CCR5 is more flexibly acceptable to heterologous NTR and UPA than GPR1, suggesting the existence of conformational differences made by the integration of multiple extracellular regions. This conformation may specifically interact with HIV-1 in a strain-dependent manner. Identification of a factor that is critical to make this conformation will contribute to understanding the mechanism of coreceptor function of GPCRs. For this, the coreceptor activity of GPR1, which is genetically distant from CCR5, will be a useful tool.

Scanning force microscopy of Escherichia coli RNA polymerase·σ54 holoenzyme complexes with DNA in buffer and in air

Scanning force microscopy (SFM) was used to visualize complexes of Escherichia coli RNA polymerase·σ54 (RNAP·σ54) and a 1036 base-pair linear DNA fragment containing the glnA promoter. In order to preserve the native hydration state of the protein-DNA complexes, the samples were injected directly into the SFM fluid cell and imaged in buffer. With this protocol, an apparent bending angle of 26(±34)° was determined for the specific complexes at the promoter. The bending angle of the unspecifically bound RNAP·σ54 showed a somewhat broader distribution of 49(±48)°, indicating the existence of conformational differences as compared to the closed complex. In about two-thirds of the closed complexes, the RNA polymerase holoenzyme was located in a lateral position with respect to the DNA and the bend of the DNA was pointing away from the protein. This conformation was consistent with the finding that for the complexes at the promoter, the apparent contour length was reduced by only about 6 nm in buffer as compared to the free DNA. From these results we conclude that in the closed complex of RNAP·σ54, the DNA was not wrapped around the polymerase, and we present a model for the trajectory of the DNA with respect to the RNA polymerase. The images acquired in buffer were compared to samples that were washed with water and then dried before imaging. Two artefacts of the washing and drying process were detected. First, extensive washing of the sample reduced the number of the specific complexes bound at the promoter (closed complex of RNAP·σ54) from about 70% to 30%. This is likely to be a result of sliding of the RNAP·σ54 holoenzyme along the DNA induced by the washing process. Second, the apparent DNA shortening of the contour length of RNAP·σ54-DNA complexes at the promoter as compared to the contour length of the free DNA was 22 nm for the dried samples as opposed to only 6 nm for the undried samples imaged in buffer. This suggests an artefact of the drying process.

Anticodon-anticodon interactions in solution: studies with yeast tRNAAsp

AbstractBakers yeast tRNAAsp has been crystallized and its crystal structure is known at high resolution (Moras et al., 1980). This tRNA possesses a quasi-complementary anticodon (GUC) and crystal studies showed that the molecules are packed in the crystalline lattice in a way involving anticodon-anticodon interactions of the Watson-Crick type. Considering the existence of conformational differences between the structure of tRNAAsp and that of tRNAphe, where such an anticodon-anticodon interaction does not exist, it has been proposed that tRNAAsp structure represents a model of a tRNA interacting with mRNA (Westhof et al., 1983). On the other hand, it is well known that two tRNA species with complementary anticodons can form stable complexes in solution (Eisinger, 1971; Grosjean et al., 1978).

Inactivation and reactivation of ribosomal subunits: Amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli

The ability of the 30 s ribosomal subunit to bind phenylalanyl-transfer RNA in the cold in response to polyuridylic acid is lost if the subunit is subjected, even transiently, to either of two treatments: (a) the removal of certain specific monovalent cations ( NH + 4, K +, Rb + or Cs +), or (b) the reduction of the Mg 2+ concentration below a critical concentration of about 2 m m. If the depleted cation is restored, the subunit reverts to an active form in a process that is greatly enhanced by heat. Thermally reactivated subunits retain full activity when rechilled, showing that the inactivation and reactivation processes involve changes, presumably conformational, in the subunit itself. Reactivation follows first-order kinetics with respect to the appearance of active subunits, with an Arrhenius activation energy of 26 kcal./mole between 30 °C and 40 °C. On storage at 0 °C, inactive 30 s subunits gradually lose the ability to be reactivated. Part of this loss is due to the oxidation of one or more sulphydryl groups and is prevented or reversed if a sulphydryl reducing agent is included in the storage or the reactivation medium, respectively. Active and inactive 30 s subunits have the same sedimentation coefficient and there is no direct evidence that they differ in conformation. However, two kinds of indirect evidence are in accord with the existence of conformational differences: (a) under appropriate conditions inactive 30 s subunits form dimers sedimenting at 50 s while active subunits do not, and active 30 s subunits associate more readily with 50 s subunits to form 70 s ribosomes; (b) inactive 30 s subunits undergo sulphydryl oxidation much more rapidly than do active ones. Although differing in certain details, the 30 s inactivation and reactivation processes are generally similar to those previously described for the 50 s subunit. Both subunits can exist in active and inactive forms which are easily and reversibly interconverted, suggesting that the structure of the functional ribosome is flexible and easily altered. The interconversions affect a number of ribosomal activities in parallel. It is possible that many previously described phenomena pertaining to ribosomal activity can be interpreted, at least in part, in terms of ribosomal inactivation and reactivation.