Automatic structure-based NMR methyl resonance assignment in large proteins.

Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28-358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.

Structural and functional insights into the interaction and targeting hub TMD0 of the polypeptide transporter TAPL.

The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.

Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome.

The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63's transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention.

NMR-based automated protein structure determination.

NMR spectra analysis for protein structure determination can now in many cases be performed by automated computational methods. This overview of the computational methods for NMR protein structure analysis presents recent automated methods for signal identification in multidimensional NMR spectra, sequence-specific resonance assignment, collection of conformational restraints, and structure calculation, as implemented in the CYANA software package. These algorithms are sufficiently reliable and integrated into one software package to enable the fully automated structure determination of proteins starting from NMR spectra without manual interventions or corrections at intermediate steps, with an accuracy of 1-2 Å backbone RMSD in comparison with manually solved reference structures.

Peak picking multidimensional NMR spectra with the contour geometry based algorithm CYPICK.

The automated identification of signals in multidimensional NMR spectra is a challenging task, complicated by signal overlap, noise, and spectral artifacts, for which no universally accepted method is available. Here, we present a new peak picking algorithm, CYPICK, that follows, as far as possible, the manual approach taken by a spectroscopist who analyzes peak patterns in contour plots of the spectrum, but is fully automated. Human visual inspection is replaced by the evaluation of geometric criteria applied to contour lines, such as local extremality, approximate circularity (after appropriate scaling of the spectrum axes), and convexity. The performance of CYPICK was evaluated for a variety of spectra from different proteins by systematic comparison with peak lists obtained by other, manual or automated, peak picking methods, as well as by analyzing the results of automated chemical shift assignment and structure calculation based on input peak lists from CYPICK. The results show that CYPICK yielded peak lists that compare in most cases favorably to those obtained by other automated peak pickers with respect to the criteria of finding a maximal number of real signals, a minimal number of artifact peaks, and maximal correctness of the chemical shift assignments and the three-dimensional structure obtained by fully automated assignment and structure calculation.

Parental dyadic coping in families of children and adolescents with type 1 diabetes.

BACKGROUND: The strains of type 1 diabetes mellitus in children and adolescents pose a challenge to the minor and his/her parents. OBJECTIVE: The objective of this study was to identify parental dyadic coping patterns and explore their relation to psychosocial and disease variables. SUBJECTS: Parents (n=44 dyads) of children/adolescents with type 1 diabetes. METHODS: Cluster analysis employing Ward's method was conducted as a multivariate classification procedure without predetermined cluster centers. RESULTS: Three parental coping clusters were identified and labeled: avoiders, negotiators, and doers. Although not reaching statistical significance, the HbA1c levels of children/adolescents with parental dyads exhibiting the negotiator coping pattern were consistently lower than the levels of children with parents classified as avoiders or doers. CONCLUSIONS: Parental dyads were distinguishable based on their dyadic coping patterns with a substantial proportion employing avoidant coping and suboptimal communication strategies. These parental dyads may benefit from minimal psychosocial intervention.

Perception of body weight status: a case control study of obese and lean children and adolescents and their parents.

BACKGROUND: The objective of this study was to investigate differences in weight perception and self-concept of obese and lean children, and to examine parents' awareness of overweight in themselves and their children. A total of 59 obese patients aged 7-17 years and 49 of their parents from a pediatric obesity out-patient clinic participated and were compared with 96 normal-weight patients and 81 of their parents from a pediatric pulmonary disease out-patient clinic. METHODS: Children's and parents' self-perception of weight, desire for weight change and weight concerns, children's belief that their desired weight can be achieved, and parents' perception of their child's weight status were assessed using single questionnaire items. Children's self-concept was measured by the Self-Perception Profile for Children. In addition, children drew pictures about themselves and their favorite activity. RESULTS: Obese patients wished to change their weight more frequently (p < 0.001) and had more weight concerns (p < 0.001). Their self-concept was significantly more negative. Physical activities were more common in their drawings than in those of normal-weight peers. Parents of obese children were more frequently overweight or obese themselves (p < 0.001). 35 of them and 73 parents of normal-weight children perceived their own weight realistically. Of the parents with overweight or obese children, 69.4% perceived their own child as overweight and 28.6% as very overweight, whereas 83% of them were obese. CONCLUSION: Children and adolescents as well as their parents recognize overweight as a health problem. In the majority, weight perception matches real body weight. Most parents at least recognize overweight in their children.

["What is important to paediatric practitioners regarding their working relationships with a university hospital?"]. / "Was ist für niedergelassene Kinderärztinnen und Kinderärzte in ihrer Arbeitsbeziehung zu einer Universitätsklinik wichtig"?

BACKGROUND: Quality management is part of Good Medical Practice. Surveys of both patient satisfaction and collaborative aspects of medical care amongst physicians in hospital and primary care are essential. METHODS: In February 2006, 92 questionnaires were distributed among office-based paediatricians in Leipzig and its suburbs. Until the reply deadline, which was in April 2006, 43 of the questionnaires had been completed and returned. The questionnaire examined attitudes of the referring physicians towards the hospital. RESULTS: Considering all answers, the Children's Hospital of the University of Leipzig received a positive rating. The results were influenced both by the age of the responding physicians and by the answer to the question of whether the respondents had previously been employed by the Children's Hospital of the University of Leipzig. CONCLUSION: This survey revealed important data on the attitudes of practitioners towards the strengths and weaknesses of the hospital. As a result, concrete measures could be implemented to enhance this co-operation with general practitioners.