PubMed · 5797532
A simple scraper for rapid and quantitative transfer of zones on thin-layer chromatograms to liquid scintillation counting vials is described.
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S Björkerud. 1969. Simple scraper for thin-layer chromatograms.. https://pubmed.ncbi.nlm.nih.gov/5797532/
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The backbone 1H, 13C, and 15N resonances of the c-Ha-Ras protein [a truncated version consisting of residues 1-171, Ras(1-171)] bound with GMPPNP (a slowly hydrolyzable analogue of GTP) were assigned and compared with those of the GDP-bound Ras(1-171). The backbone amide resonances of amino acid residues 10-13, 21, 31-39, 57-64, and 71 of Ras(1-171).GMPPNP, but not those of Ras(1-171).GDP, were extremely broadened, whereas other residues of Ras(1-171).GMPPNP exhibited amide resonances nearly as sharp as those of Ras(1-171). GDP. The residues exhibiting the extreme broadening, except for residues 21 and 71, are localized in three functional loop regions [loops L1, L2 (switch I), and L4 (switch II)], which are involved in hydrolysis of GTP and interactions with other proteins. From the temperature and magnetic field strength dependencies of the backbone amide resonance intensities, the extreme broadening was ascribed to the exchange at an intermediate rate on the NMR time scale. It was shown that the Ras(1-171) protein bound with GTP or GTPgammaS (another slowly hydrolyzable analogue of GTP) exhibits the same type of broadening. Therefore, it is a characteristic feature of the GTP-bound form of Ras that the L1, L2, and L4 loop regions, but not other regions, are in a rather slow interconversion between two or more stable conformers. This phenomenon, termed a "regional polysterism", of these loop regions may be related with their multifunctionality: the GTP-dependent interactions with several downstream target groups such as the Raf and RalGDS families and also with the GTPase activating protein (GAP) family. In fact, the binding of Ras(1-171).GMPPNP with the Ras-binding domain (residues 51-131) of c-Raf-1 was shown to eliminate the regional polysterism nearly completely. It was indicated, therefore, that each target/regulator selects its appropriate conformer among those presented by the "polysteric" binding interface of Ras. As the downstream target groups exhibit no apparent sequence homology to each other, it is possible that one target group prefers a conformer different from that preferred by another group. The involvement of loop L1 in the regional polysterism might suggest that the negative regulators, GAPs, bind to the polysteric binding interface (loops L2 and L4) of Ras and cooperatively select a conformer suitable for transition of the GTPase catalytic center, involving loops L1 and L4, into the highly active state.
Conformations of G-CSF and the extracellular domain of its receptor as well as their complex have been investigated by employing isotope-edited FTIR spectroscopy. To determine unambiguously the protein conformations of G-CSF and the receptor in the complex, we have prepared uniformly 13C/15N isotope labeled G-CSF to resolve its amide I' band from that of the receptor in the IR spectrum of the complex. By comparing the IR spectra of the isotope-labeled G-CSF and the receptor with that of the complex, we have provided spectral evidence that the AB loop region involving the unique 310 helix segment of G-CSF likely undergoes a conformational change to a regular alpha-helix upon binding to the receptor. The IR data also indicate a possible minor increase in alpha-helical conformation for the receptor in the complex. Furthermore, FTIR spectra of G-CSF, the receptor, and their complex demonstrate clearly that protein conformations of both G-CSF and the receptor have been dramatically stabilized by complex formation. Specifically, the melting transition (Tm value) of the alpha-helix in G-CSF is increased by nearly 30 degrees C and that of the beta-strand in the receptor by nearly 15 degrees C in the G-CSF/receptor complex. We estimate from the current FTIR data that the native conformations of approximately 15% of all receptor residues are stabilized by G-CSF binding. On the other hand, the entire alpha-helical content of G-CSF appears to be stabilized in the complex. Together, these results indicate that formation of the ligand/receptor complex results in not only conformational changes in the receptor but also significant structural changes in the ligand. This adds insight to the general consensus that binding of ligand to cytokine receptors induces mostly structural changes in the receptor which lead to receptor oligomerization and signal transduction. The current data also suggest a possible physiological role of the 310 helix present in G-CSF for its receptor binding activity.
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