Biomedical subjects
Publications and source records attributed to R Hilgenfeld.
Both recombinant blood coagulation factor XIII alpha-chain and factor XIII isolated from human placenta have been crystallized using a novel robotic system for the automatic screening of crystallization conditions. The monoclinic and orthorhombic crystals obtained are suitable for X-ray analysis.
Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation.
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The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3.2 A resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0.857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of alpha-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin. Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.
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Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT). All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pulT mutations cluster in the three-domain junction interface of EF-Tu. GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tRNA binding. Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.