Browsing by Subject "Amino terminal sequence"

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Open Access
Functional role of the noncatalytic domains of elongation factor tu in the interactions with ligands
(1998) Cetin, R.; Anborgh, P. H.; Cool, R. H.; Parmeggiani, A.
Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(AM) and EF-Tu(δC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N- terminal domain, they had lost a typical feature of EF-Tu, the ≤ 100 times stronger affinity for GDP than for GTP. EF-Tu(ΔM) and EF-Tu(AC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(ΔC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was ≤90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF- Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(ΔC)·GTP, showing the same kind of effect as on EF-Tu·GTP, but they were little active on EF-Tu(ΔM)·GTP. Like EF-Tu(ΔC)·GTP, EF-Tu(ΔM)·GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.
Open Access
A new set of monoclonal antibodies directed to proline-rich and central regions of p53
(Mary Ann Liebert, Inc., 2004) Voeltzel, T.; Morel, A. P.; Rostan, M. C.; Ji, J.; Chiodino, C.; Ponchel, F.; Vigouroux, J.; De Fromentel, C. C.; Soussi, T.; Ozturk, M.
The p53 protein can adopt several conformations in cells - "latent," "active," or mutant - depending on cellular stress or mutations of the TP53 gene. Today, only a few antibodies discriminating these conformations are available. We produced three new anti-p53 monoclonal antibodies (MAbs) directed against epitopes of human p53. The H53C1 MAb recognizes an epitope located at the N-terminal part of the central region of p53 and can discriminate mutant from wild-type conformation. The H53C2 and H53C3 MAbs are against different epitopes within the proline-rich region of p53. Moreover, the H53C2 epitope is located in the second negative regulatory domain of p53 between residues 80 and 93. These MAbs can be used as new tools to study and modulate the cellular functions of p53.