Biomedical subjects

Thomas Huber

Publications and source records attributed to Thomas Huber.

27 records · Page 2Linked to original sources

PubMed2004

Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase.

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.

Amino Acid Motifs↗

PubMed2004

Membrane model for the G-protein-coupled receptor rhodopsin: hydrophobic interface and dynamical structure.

Rhodopsin is the only member of the pharmacologically important superfamily of G-protein-coupled receptors with a known structure at atomic resolution. A molecular dynamics model of rhodopsin in a POPC phospholipid bilayer was simulated for 15 ns, revealing a conformation significantly different from the recent crystal structures. The structure of the bilayer compared with a protein-free POPC control indicated hydrophobic matching with the nonpolar interface of the receptor, in agreement with deuterium NMR experiments. A new generalized molecular surface method, based on a three-dimensional Voronoi cell construction for atoms with different radii, was developed to quantify cross-sectional area profiles for the protein, lipid acyl chains and headgroups, and water. Thus, it was possible to investigate the bilayer deformation due to curvature of the individual lipid monolayers. Moreover, the generalized molecular surface derived hydrophobic interface allowed benchmarking of the hydropathy sequence analysis, an important structural genomics tool. Five water molecules diffused into internal hydration sites during the simulation, yielding a total of 12 internal waters. The cytoplasmic loops and the C-terminal tail, containing the G-protein recognition and protein sorting sequences, exhibited a high mobility, in marked contrast to the extracellular and transmembrane domains. The proposed functional coupling of the highly conserved ERY motif to the lipid-water interface via the cytoplasmic loops provides insight into lipid effects on G-protein-coupled receptor activation in terms of a flexible surface model, involving the spontaneous monolayer curvature.

Animals↗

PubMed2004

Triterpenes from Maesopsis eminii.

Two pentacyclic triterpenes, 1alpha,3beta-dihydroxybauer-7-en-28-oic acid (1) and 3beta-hydroxybauer-7-en-28-oic acid (2), together with sitosterol-3-beta-O-d-glucopyranoside and stigmasterol have been isolated from the bark of the plant Maesopsis eminii. Their structures have been elucidated by spectroscopic methods. One of the triterpenes (1) is new, and its structure was confirmed by X-ray crystallographic analysis. This new triterpene displayed moderate antibacterial activity against Bacillus subtilis ATCC 6633.

Anti-Bacterial Agents↗

PubMed2003

Biophysical properties of human antibody variable domains.

There are great demands on the stability, expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V(H)) and light (V(L)) germline family consensus domains and then a systematic series of V(H)-V(L) combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V(H) and seven V(L) domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL). The isolated V(H) and V(L) domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V(lambda) domains show high levels of stability, even though V(lambda) domains are surprisingly unstable by themselves. This is due to a strong interaction with the V(H) domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design.

Amino Acid Sequence↗

PubMed2003

Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases.

A significant number of chimeric 16S rDNA sequences of diverse origin were identified in the public databases by partial treeing analysis. This suggests that chimeric sequences, representing phylogenetically novel non-existent organisms, are routinely being overlooked in molecular phylogenetic surveys despite a general awareness of PCR-generated artefacts amongst researchers.

Chimera↗

PubMed2003

Systematic characterization of the zinc-finger-containing proteins in the mouse transcriptome.

Zinc-finger-containing proteins can be classified into evolutionary and functionally divergent protein families that share one or more domains in which a zinc ion is tetrahedrally coordinated by cysteines and histidines. The zinc finger domain defines one of the largest protein superfamilies in mammalian genomes;46 different conserved zinc finger domains are listed in InterPro (http://www.ebi.ac.uk/InterPro). Zinc finger proteins can bind to DNA, RNA, other proteins, or lipids as a modular domain in combination with other conserved structures. Owing to this combinatorial diversity, different members of zinc finger superfamilies contribute to many distinct cellular processes, including transcriptional regulation, mRNA stability and processing, and protein turnover. Accordingly, mutations of zinc finger genes lead to aberrations in a broad spectrum of biological processes such as development, differentiation, apoptosis, and immunological responses. This study provides the first comprehensive classification of zinc finger proteins in a mammalian transcriptome. Specific detailed analysis of the SP/Krüppel-like factors and the E3 ubiquitin-ligase RING-H2 families illustrates the importance of such an analysis for a more comprehensive functional classification of large protein families. We describe the characterization of a new family of C2H2 zinc-finger-containing proteins and a new conserved domain characteristic of this family, the identification and characterization of Sp8, a new member of the Sp family of transcriptional regulators, and the identification of five new RING-H2 proteins.

Alternative Splicing↗

PubMed2003

Phosphoregulators: protein kinases and protein phosphatases of mouse.

With the completion of the human and mouse genome sequences, the task now turns to identifying their encoded transcripts and assigning gene function. In this study, we have undertaken a computational approach to identify and classify all of the protein kinases and phosphatases present in the mouse gene complement. A nonredundant set of these sequences was produced by mining Ensembl gene predictions and publicly available cDNA sequences with a panel of InterPro domains. This approach identified 561 candidate protein kinases and 162 candidate protein phosphatases. This cohort was then analyzed using TribeMCL protein sequence similarity clustering followed by CLUSTALV alignment and hierarchical tree generation. This approach allowed us to (1) distinguish between true members of the protein kinase and phosphatase families and enzymes of related biochemistry, (2) determine the structure of the families, and (3) suggest functions for previously uncharacterized members. The classifications obtained by this approach were in good agreement with previous schemes and allowed us to demonstrate domain associations with a number of clusters. Finally, we comment on the complementary nature of cDNA and genome-based gene detection and the impact of the FANTOM2 transcriptome project.

Animals↗

PubMed2002

Structure of docosahexaenoic acid-containing phospholipid bilayers as studied by (2)H NMR and molecular dynamics simulations.

Polyunsaturated phospholipids are known to be important with regard to the biological functions of essential fatty acids, for example, involving neural tissues such as the brain and retina. Here we have employed two complementary structural methods for the study of polyunsaturated bilayer lipids, viz. deuterium ((2)H) NMR spectroscopy and molecular dynamics (MD) computer simulations. Our research constitutes one of the first applications of all-atom MD simulations to polyunsaturated lipids containing docosahexaenoic acid (DHA; 22:6 cis-Delta(4,7,10,13,16,19)). Structural features of the highly unsaturated, mixed-chain phospholipid, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), have been studied in the liquid-crystalline (L(alpha)) state and compared to the less unsaturated homolog, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The (2)H NMR spectra of polyunsaturated bilayers are dramatically different from those of less unsaturated phospholipid bilayers. We show how use of MD simulations can aid in interpreting the complex (2)H NMR spectra of polyunsaturated bilayers, in conjunction with electron density profiles determined from small-angle X-ray diffraction studies. This work clearly demonstrates preferred helical and angle-iron conformations of the polyunsaturated chains in liquid-crystalline bilayers, which favor chain extension while maintaining bilayer flexibility. The presence of relatively long, extended fatty acyl chains may be important for solvating the hydrophobic surfaces of integral membrane proteins, such as rhodopsin. In addition, the polyallylic DHA chains have a tendency to adopt back-bended (hairpin-like) structures, which increase the interfacial area per lipid. Finally, the material properties have been analyzed in terms of the response of the bilayer to mechanical stress. Simulated bilayers of phospholipids containing docosahexaenoic acid were less sensitive to the applied surface tension than were saturated phospholipids, possibly implying a decrease in membrane elasticity (area elastic modulus, bending rigidity). The above features distinguish DHA-containing lipids from saturated or monounsaturated lipids and may be important for their biological modes of action.

Computer Simulation↗

PubMed2002

A solid-state NMR study of phospholipid-cholesterol interactions: sphingomyelin-cholesterol binary systems.

We used solid-state NMR techniques to probe the interactions of cholesterol (Chol) with bovine brain sphingomyelin (SM) and for comparison of the interactions of Chol with dipalmitoylphosphatidylcholine (DPPC), which has a similar gel-to-liquid crystalline transition temperature. (1)H-, (31)P-, and (13)C-MASNMR yielded high-resolution spectra from multilamellar dispersions of unlabeled brain SM and Chol for analysis of chemical shifts and linewidths. In addition, (2)H-NMR spectra of oriented lipid membranes with specific deuterium labels gave information about membrane ordering and mobility. Chol disrupted the gel-phase of pure SM and increased acyl chain ordering in the liquid crystalline phase. As inferred from (13)C chemical shifts, the boundaries between the ordered and disordered liquid crystalline phases (L and L) were similar for SM and DPPC. The solubility limit of Chol in SM was ~50 mol %, the same value as previously reported for DPPC membranes. We found no evidence for specific H-bonding between Chol and the amide group of SM. The order parameters of a probe molecule, d31-sn1-DPPC, in SM were slightly higher than in DPPC for all carbons except the terminal groups at 30 mol % but were not significantly different at 5 and 60 mol % Chol. These studies show a general similarity with some subtle differences in the way Chol interacts with DPPC and SM. In the environment of a typical biomembrane, the higher proportion of saturated fatty acyl chains in SM compared to other phospholipids may be the most significant factor influencing interactions with Chol.

1,2-Dipalmitoylphosphatidylcholine↗