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Biomedical subjects

G Wagner

Publications and source records attributed to G Wagner.

At least 127 records · Page 7Linked to original sources
PubMed1999

One of two unstructured domains of Ii becomes ordered in complexes with MHC class II molecules.

We studied the role of the invariant chain (Ii) protein's structure in its ability to form complexes with major histocompatibility complex class II molecules. Multidimensional nuclear magnetic resonance experiments demonstrated that Ii contains two unstructured, flexible domains: a 39 residue sequence that contains a region (CLIP) critical for Ii/class II complex formation and becomes rapidly ordered when Ii/class II complexes are assembled, and a 30 residue sequence that contains the insertion point for a protease inhibitor domain included in an alternative splice form of Ii. Mobility of these domains guarantees accessibility to CLIP and the inhibitor insert, and ordering of the CLIP-containing domain may provide protection against proteolysis and contribute, along with Ii's compact 118-192 domain, to allotype-independent class II binding.

Amino Acid Sequence↗
PubMed1999

Efficient side-chain and backbone assignment in large proteins: application to tGCN5.

In determining the structure of large proteins by NMR, it would be desirable to obtain complete backbone, side-chain, and NOE assignments efficiently, with a minimum number of experiments and samples. Although new strategies have made backbone assignment highly efficient, side-chain assignment has remained more difficult. Faced with the task of assigning side-chains in a protein with poor relaxation properties, the Tetrahymena histone acetyltransferase tGCN5, we have developed an assignment strategy that would provide complete side-chain assignments in cases where fast 13C transverse relaxation causes HCCH-TOCSY experiments to fail. Using the strategy presented here, the majority of aliphatic side-chain proton and carbon resonances can be efficiently obtained using optimized H(CC-CO)NH-TOCSY and (H)C(C-CO)NH-TOCSY experiments on a partially deuterated protein sample. Assignments can be completed readily using additional information from a 13C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOSCY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.

Acetyltransferases↗
PubMed1999

Application of automated NOE assignment to three-dimensional structure refinement of a 28 kDa single-chain T cell receptor.

An automated procedure for NOE assignment and three-dimensional structure refinement is presented. The input to the procedure consists of (1) an ensemble of preliminary protein NMR structures, (2) partial sequence-specific assignments for the protein and (3) the positions and volumes of unassigned NOESY cross peaks. Chemical shifts for unassigned side chain protons are predicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment and structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 408-422]. ARIA is optimized for the task of structure refinement of larger proteins. Errors are filtered to ensure that sequence-specific assignments are reliable. The procedure is applied to the 27.8 kDa single-chain T cell receptor (scTCR). Preliminary NMR structures, nearly complete backbone assignments, partial assignments of side chain protons and more than 1300 unassigned NOESY cross peaks are input. Using the procedure, the resonant frequencies of more than 40 additional side chain protons are assigned. Over 400 new NOE cross peaks are assigned unambiguously. Distances derived from the automatically assigned NOEs improve the precision and quality of calculated scTCR structures. In the refined structures, a hydrophobic cluster of side chains on the scTCR surface that binds major histocompatibility complex (MHC)/antigen is revealed. It is composed of the side chains of residues from three loops and stabilizes the conformation of residues that interact with MHC.

Histocompatibility Antigens Class I↗
PubMed1999

The GYF domain is a novel structural fold that is involved in lymphoid signaling through proline-rich sequences.

T cell activation through the CD2 cell surface receptor is transmitted by proline-rich sequences within its cytoplasmic tail. A membrane-proximal proline-rich tandem repeat, involved in cytokine production, is recognized by the intracellular CD2 binding protein CD2BP2. We solved the solution structure of the CD2 binding domain of CD2BP2, which we name the glycine-tyrosine-phenylalanine (GYF) domain. The GYF sequence is part of a structurally unique bulge-helix-bulge motif that constitutes the major binding site for the CD2 tail. A hydrophobic surface patch is created by motif residues that are highly conserved among a variety of proteins from diverse eukaryotic species. Thus, the architecture of the GYF domain may be widely used in protein-protein associations.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Solution structure of the hRPABC14.4 subunit of human RNA polymerases.

The protein hRPABC14.4 is an essential subunit of human RNA polymerases I, II, and III and is required for the transcription of all human nuclear genes. The structure of hRPABC14.4 was determined by nuclear magnetic resonance spectroscopy. The protein fold comprises a highly conserved central domain forming two antiparallel alpha-helices flanked by the less conserved N- and C-terminal regions forming a five-stranded beta-sandwich. Amino acids from the two helices participate in the generation of a hydrophobic surface area which is conserved in all eukaryotic and archaeal homologous subunits, and likely constitutes a critical macromolecular interaction interface. The hRPABC14.4 structure accounts for mutagenesis results in Saccharomyces cerevisiae and provides a structural working model for elucidating the role of this subunit in the molecular architecture and function of the human nuclear RNA polymerases.

Amino Acid Sequence↗
PubMed1999

Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor.

Using NMR spectroscopy, we determined the solution structure of a single-chain T-cell receptor (scTCR) derived from the major histocompatibility complex (MHC) class II-restricted D10 TCR. The conformations of complementarity-determining regions (CDRs) 3beta and 1alpha and surface properties of 2alpha are different from those of related class I-restricted TCRs. We infer a conserved orientation for TCR V(alpha) domains in complexes with both class I and II MHC-peptide ligands, which implies that small structural variations in V(alpha) confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond time scale) provides insight into immune recognition and selection mechanisms.

Amino Acid Sequence↗
PubMed1999

[Clinical and electrophysiologic studies of facial nerve function after hypoglossal-facial nerve anastomosis].

BACKGROUND: The functional and cosmetic results after hypoglossal-facial nerve anastomosis (HFA) have primarily been described using a subjective grading system of good, fair, or poor. To obtain more objective results, the postoperative mimic function was investigated using a combination of clinical and electrophysiological methods. METHODS: A total of 29 patients with hypoglossal-facial anastomosis were evaluated using a standardized questionnaire and the Stennert's paralysis index as clinical scoring system and electroneurography (NMG/ENoG) and electromyography (EMG) as electrophysiological assessments. RESULTS: Twenty-six of 29 patients (89%) judged their own facial function as very good or good, and three as poor. Under resting tone conditions, no patient showed a difference between palpebral fissures (> 3 mm), an ectropium, a substantial loss of nasolabial fold, or a drop of angulus oris (> 3 mm). With attempted movement, the lid closure was complete in 76% of patients, and the cornea was lid-covered in 100%. Sixty-seven percent of the patients were able to whistle. The second upper incisor was visible (full width) in 19%. No patient could frown sufficiently or expose the upper and lower canine teeth. The mean Stennert's score decreased from 9 to 4 after surgery. The relative amplitudes of the NMGs were increased in patients with low clinical indices. Patients with a complete eye closure showed a higher density of EMG patterns in their orbicularis oculi muscles. No significant differences for clinical or electrophysiological parameters could be observed based on age, sex, or time point of operation. CONCLUSION: Combining the Stennert's paralysis index with NMG and EMG allows a more objective assessment of functional and cosmetic results after HFA.

Adolescent↗
PubMed1999

Exclusion of the candidate genes ACE and Bcl-2 for six families with nephronophthisis not linked to the NPH1 locus.

BACKGROUND: Nephronophthisis (NPH) is an autosomal recessively transmitted kidney disease, characterized by cyst formation at the cortico-medullary junction, and a sclerosing tubulointerstitial nephropathy. Juvenile nephronophthisis (NPH1) is the most common genetic cause of renal failure in children and maps to chromosome 2q12-q13. The responsible gene NPHP1 has been identified and encodes for nephrocystin. Not all families with NPH demonstrate linkage to that locus. METHODS: We studied six families with NPH without linkage to the NPH1 locus. In order to attempt identification of a new causative gene, the candidate genes ACE (angiotensin converting enzyme) and Bcl-2 (B cell leukaemia/lymphoma 2 gene) originating from mouse models, were examined. For the six families highly polymorphic microsatellites covering the whole candidate gene regions were haplotyped and linkage analysis was performed. RESULTS: Haplotype analyses of all families examined were incompatible with linkage of the disease status to ACE or Bcl-2. Linkage analysis excluded both candidate gene regions with a LOD-score of < -2. CONCLUSIONS: This study excluded the candidate genes ACE and Bcl-2 for NPH. Additional linkage studies need to be performed in order to identify further genes responsible for nephronophthisis.

Adolescent↗
PubMed1999

Dynamics of water coning.

Water beneath a layer of oil in a producing reservoir may rise and form a bell-shaped cone in the vicinity of the well. We discuss how the dynamics of cone formation in two dimensions depends on the gravitational contrast, the interfacial tension, and the flow rate of oil. For a constant flow rate below a critical rate Q(c), stable cones are formed. At rates above Q(c), two dynamical regimes are expected. These are slow initial formation and fast breakthrough to the well. Quasi-two-dimensional transparent porous models were used to perform cone formation experiments. Effective acceleration due to gravity was systematically varied. The experiments were simulated using a stochastic model based on invasion percolation in which capillary forces were explicitly taken into account. We find agreement between experiments and simulations, and consistency with the theoretical predictions.

Journal Article↗
PubMed1999

Extended Eden model reproduces growth of an acellular slime mold.

A stochastic growth model was used to simulate the growth of the acellular slime mold Physarum polycephalum on substrates where the nutrients were confined in separate drops. Growth of Physarum on such substrates was previously studied experimentally and found to produce a range of different growth patterns [Phys. Rev. E 57, 941 (1998)]. The model represented the aging of cluster sites and differed from the original Eden model in that the occupation probability of perimeter sites depended on the time of occupation of adjacent cluster sites. This feature led to a bias in the selection of growth directions. A moderate degree of persistence was found to be crucial to reproduce the biological growth patterns under various conditions. Persistence in growth combined quick propagation in heterogeneous environments with a high probability of locating sources of nutrients.

Algorithms↗
PubMed1998

Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation.

An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also found in several yeast and Caenorhabditis elegans proteins of unknown function. In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains regulates protein-protein interactions.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Structure of a trimeric domain of the MHC class II-associated chaperonin and targeting protein Ii.

The invariant chain (Ii) plays a critical role in MHC class II antigen processing by stabilizing peptide-free class II alphabeta heterodimers in a nonameric (alphabetaIi)3 complex soon after their synthesis and directing transport of the complex from the endoplasmic reticulum to compartments where peptide loading of class II takes place. Loading progresses following Ii proteolysis and via an intermediate complex of MHC class II with an Ii-derived peptide, CLIP. CLIP is substituted by exogenous peptidic fragments in an exchange reaction catalyzed by HLA-DM. The CLIP region of Ii, roughly residues 81-104, is one of two segments shown to interact with class II HLA-DR molecules. The other segment, Ii 118-216, is C-terminal to CLIP, mediates trimerization of the ectodomain of Ii and interferes with DM/class II binding. Here we report the three-dimensional structure of this trimeric domain, determined by nuclear magnetic resonance (NMR) studies of a 27 kDa trimer of human Ii 118-192. The cylindrical shape of the molecule and the mapping of conserved residues delimit surfaces which may be important for interactions between Ii and class II molecules.

Amino Acid Sequence↗
PubMed1998

Folding kinetics of villin 14T, a protein domain with a central beta-sheet and two hydrophobic cores.

The thermodynamics and kinetics of folding are characterized for villin 14T, a 126-residue protein domain. Equilibrium fluorescence measurements reveal that villin 14T unfolds and refolds reversibly. The folding kinetics was monitored using stopped-flow with fluorescence and quenched-flow with NMR and mass spectrometry. Unfolding occurs in a single-exponential phase in the stopped-flow experiments, and about 75% of the total amplitude is recovered in the fast phase of refolding. The remaining 25% of the amplitude probably represents trapping in cis-trans proline isomerization pathways. At 25 degreesC, the stability estimate obtained by extrapolation from the transition region of the stopped-flow chevron matches the stability value from equilibrium urea titrations (DeltaG = 9.7 kcal/mol, m value = 2.2 kcal mol-1 M-1). At low final urea concentrations, however, the refolding kinetics deviates from the two-state model, indicating the formation of an intermediate. Under these conditions, quenched-flow followed by NMR and mass spectrometry show no detectable hydrogen-bonded intermediate in the fast refolding phase. In contrast, agreement is observed between the equilibrium and kinetic estimates of stability at 37 degreesC (DeltaG = 6.0 kcal/mol, m value = 1.6 kcal mol-1 M-1), at all observed urea concentrations, demonstrating apparent two-state folding at this temperature. This result shows that the two-state folding model, previously applied to small domains with single, central hydrophobic cores, can also describe the folding of a larger domain with multiple core structures.

Animals↗
PubMed1998

Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment.

Apoptosis requires recruitment of caspases by receptor-associated adaptors through homophilic interactions between the CARDs (caspase recruitment domains) of adaptor proteins and prodomains of caspases. We have solved the CARD structure of the RAIDD adaptor protein that recruits ICH-1/caspase-2. It consists of six tightly packed helices arranged in a topology homologous to the Fas death domain. The surface contains a basic and an acidic patch on opposite sides. This polarity is conserved in the ICH-1 CARD as indicated by homology modeling. Mutagenesis data suggest that these patches mediate CARD/CARD interaction between RAIDD and ICH-1. Subsequent modeling of the CARDs of Apaf-1 and caspase-9, as well as Ced-4 and Ced-3, showed that the basic/acidic surface polarity is highly conserved, suggesting a general mode for CARD/CARD interaction.

Amino Acid Sequence↗