The fast Fourier transformation has been the gold standard for transforming data from time to frequency domain in many spectroscopic methods, including NMR. While reliable, it has as a drawback that it requires a grid of uniformly sampled data points. This needs very long measuring times for sampling in multidimensional experiments in all indirect dimensions uniformly and even does not allow reaching optimal evolution times that would match the resolution power of modern high-field instruments. Thus, many alternative sampling and transformation schemes have been proposed. Their common challenges are the suppression of the artifacts due to the non-uniformity of the sampling schedules, the preservation of the relative signal amplitudes, and the computing time needed for spectra reconstruction. Here we present a fast implementation of the Iterative Soft Thresholding approach that can reconstruct high-resolution non-uniformly sampled NMR data up to four dimensions within a few hours and make routine reconstruction of high-resolution NUS 3D and 4D spectra convenient. We include a graphical user interface for generating sampling schedules with the Poisson-Gap method and an estimation of optimal evolution times based on molecular properties. The performance of the approach is demonstrated with the reconstruction of non-uniformly sampled medium and high-resolution 3D and 4D protein spectra acquired with sampling densities as low as 0.8%. The method presented here facilitates acquisition, reconstruction and use of multidimensional NMR spectra at otherwise unreachable spectral resolution in indirect dimensions.

SUMMARY Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2 and eIF5. The carboxyl-terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and find that they are partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. By abrogating eIF5-CTD binding to eIF2β, genetic and biochemical evidence indicate that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC. This study provides mechanistic insight into the novel role of eIF5-CTD’s dynamic interplay with eIF1 and eIF2β in switching PICs from an open to closed state at start codons.

The human Activator-Recruited Cofactor (ARC)/Mediator co-activator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 (ARC92) subunit is a critical target of the potent Herpes simplex 1 viral transcriptional activator VP16. Here, we determine the solution structure of the MED25 VP16-binding domain (VBD), and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a β-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of over-expressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms.

In analogy to the cystine knots present in natural collagens, a simplified disulfide cross-link was used to analyse the conformational effects of a C-terminal artificial cystine knot on the folding of collagenous peptides consisting of solely (Pro-Hyp-Gly) repeating units. Assembly of the alpha chains into a heterotrimer by previously applied regioselective disulfide-bridging strategies failed because of the high tendency of (Pro-Hyp-Gly)(5) peptides to self-associate and form homotrimers. Only when side-chain-protected peptides were used, for example in the Hyp(tBu) form, and a new protection scheme was adopted, selective interchain-disulfide cross-linking into the heterotrimer in organic solvents was successful. This unexpected strong effect of the conformational properties on the efficiency of well-established reactions was further supported by replacing the Hyp residues with (4S)-fluoroproline, which is known to destabilise triple-helical structures. With the related [Pro-(4S)-FPro-Gly](5) peptides, assembly of the heterotrimer in aqueous solution proceeded in a satisfactory manner. Both the intermediates and the final fluorinated heterotrimer are fully unfolded in aqueous solution even at 4 degrees C. Conversely, the disulfide-crossbridged (Pro-Hyp-Gly)(5) heterotrimer forms a very stable triple helix. The observation that thermal unfolding leads to scrambling of the disulfide bridges was unexpected. Although NMR experiments support an extension of the triple helix into the cystine knot, thermolysis is not associated with the unfolding process. In fact, the unstructured fluorinated trimer undergoes an equally facile thermodegradation associated with the intrinsic tendency of unsymmetrical disulfides to disproportionate into symmetrical disulfides under favourable conditions. The experimental results obtained with the model peptides fully support the role of triple-helix nucleation and stabilisation by the artificial cystine knot as previously suggested for the natural cystine knots in collagens.