SignificanceThe p63 gene encodes a master regulator of epidermal development and function. Specific mutations in p63 are causative of a life-threatening disorder mainly characterized by severe skin erosions and cleft palate. Little is known about the mechanisms underlying disease pathology and possible treatments. Based on biochemical studies, genetic mouse models, and functional assays, we demonstrate that these mutations cause p63 protein misfolding and aggregation. Protein aggregation lead to reduced DNA binding and impaired transcriptional activity. Importantly, genetic modifications of p63 that abolish aggregation of the mutant proteins rescue its function, revealing that ankyloblepharon-ectodermal defects-cleft lip/palate syndrome is a protein aggregation disorder and opening avenues for therapeutic intervention.

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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.

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.

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.

Isotope-labeled methyl groups provide NMR probes that can be observed in very large, otherwise deuterated systems and enable investigations of protein structure, dynamics and mechanisms.However, the assignment of resonances to specific methyls in the protein is expensive and timeconsuming, which limits the use of methyl-based NMR for large proteins. To resolve this bottleneck, methyl assignment methods have been developed. However, these remain limited regarding complete automation, computational feasibility, and/or the extent and accuracy of the assignments. 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 was applied to five proteins (28-358 kDa) comprising a total of 708 isotopelabeled methyl groups, of which 674 had manually determined 1 H/ 13 C reference assignments and 614 showed cross peaks in the available NOESY peak lists. MethylFLYA confidently assigned 488 methyl groups, i.e. 79% of those with NOESY data. Of these, 460 agreed with the reference, 5 were different, and 23 concerned methyls without reference assignment. For high-quality NOESY spectra, automatic NOESY peak picking followed by resonance assignment with MethylFLYA can yield results comparable to those obtained from manually prepared peak lists, indicating the feasibility of unbiased, fully automatic methyl resonance assignment starting directly from the NMR spectra. Overall, MethylFLYA assigns significantly more methyl groups than other algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h, and flexibility to handle arbitrary isotope labeling patterns and include data from other types of NMR spectra.

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