RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell‐specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences (‘zipcodes’) within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA‐binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p's C‐terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N‐terminal coiled‐coil domain of She3p is required to form an RNA‐independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA–motor protein adapters.

It has been demonstrated that the fragmentation scheme of our adjustable density matrix assembler (ADMA) approach for the quantum chemical calculations of very large systems is well-suited to calculate NMR chemical shifts of proteins [Frank et al. Proteins 2011, 79, 2189–2202]. The systematic investigation performed here on the influences of the level of theory, basis set size, inclusion or exclusion of an implicit solvent model, and the use of partial charges to describe additional parts of the macromolecule on the accuracy of NMR chemical shifts demonstrates that using a valence triple-ζ basis set leads to large improvement compared to the results given in the previous publication. Additionally, moving from the B3LYP to the mPW1PW91 density functional and including partial charges and implicit solvents gave the best results with mean absolute errors of 0.44 ppm for hydrogen atoms excluding HN atoms and between 1.53 and 3.44 ppm for carbon atoms depending on the size and also on the accuracy of the protein structure. Polar hydrogen and nitrogen atoms are more difficult to predict. For the first, explicit hydrogen bonds to the solvents need to be included and, for the latter, going beyond DFT to post-Hartree–Fock methods like MP2 is probably required. Even if empirical methods like SHIFTX+ show similar performance, our calculations give for the first time very reliable chemical shifts that can also be used for complexes of proteins with small-molecule ligands or DNA/RNA. Therefore, taking advantage of its ab initio nature, our approach opens new fields of application that would otherwise be largely inaccessible due to insufficient availability of data for empirical parametrization.

Fragment-based quantum chemical calculations are able to accurately calculate NMR chemical shifts even for very large molecules like proteins. But even with systematic optimization of the level of theory and basis sets as well as the use of implicit solvents models, some nuclei like polar protons and nitrogens suffer from poor predictions. Two properties of the real system, strongly influencing the experimental chemical shifts but almost always neglected in the calculations, will be discussed here in great detail: (1) conformational averaging and (2) interactions with first-shell solvent molecules. Classical molecular dynamics simulations in explicit water were carried out for obtaining a representative ensemble including the arrangement of neighboring solvent molecules, which was then subjected to quantum chemical calculations. We could demonstrate with the small test system N-methyl acetamide (NMA) that the calculated chemical shifts show immense variations of up to 6 ppm and 50 ppm for protons and nitrogens, respectively, depending on the snapshot taken from a classical molecular dynamics simulation. Applying the same approach to the HA2 domain of the influenza virus glycoprotein hemagglutinin, a 32-amino-acid-long polypeptide, and comparing averaged values to the experiment, chemical shifts of nonpolar protons and carbon atoms in proteins were calculated with unprecedented accuracy. Additionally, the mean absolute error could be reduced by a factor of 2.43 for polar protons, and reasonable correlations were obtained for nitrogen and carbonyl carbon in contrast to all other studies published so far.

Despite the many protein structures solved successfully by nuclear magnetic resonance (NMR) spectroscopy, quality control of NMR structures is still by far not as well established and standardized as in crystallography. Therefore, there is still the need for new, independent, and unbiased evaluation tools to identify problematic parts and in the best case also to give guidelines that how to fix them. We present here, quantum chemical calculations of NMR chemical shifts for many proteins based on our fragment-based quantum chemical method: the adjustable density matrix assembler (ADMA). These results show that (13)C chemical shifts of reasonable accuracy can be obtained that can already provide a powerful measure for the structure validation. (1)H and even more (15)N chemical shifts deviate more strongly from experiment due to the insufficient treatment of solvent effects and conformational averaging.

Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.

Various 1-(1-hydroxyalkyl) paraconyl alcohols are important signaling molecules within antibiotics production in Streptomyces sp. Intending developing a flexible convergent chemical synthesis of such butanolides, a zwitterionic aza-Claisen rearrangement was chosen as reliable strategy generating the central stereotriad. Reaction of enantiopure N-allyl pyrrolidines and 4-phenylbutenoic acid fluoride delivered defined configured amides displaying the 2,3,1' stereotriads. The configuration was determined by the allyl alcohol moiety indicating a complete remote stereo control. Amide removal by iodolactonization and proceeding reductions, halocyclization and elimination gave key alkylidene tetrahydrofuran derivatives. Stepwise degradation of the olefins through ozonolysis, reductive workup and protecting group removal delivered both enantiomers of the target Streptomyces coelicolor butanolide 5.

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(13)C chemical shifts of alkynes, published to date, were computed at the DFT (B3LYP/6-311G*) level of theory and compared with the experimental delta values, and the agreement was employed as a measure of quality for the underlying structures. For the corresponding global minima structures, thus obtained, the occupation quotients of antibonding pi* and bonding pi orbitals (pi*(C[triple bond]C)/pi(C[triple bond]C)) and the bond lengths (d(C[triple bond]C)) of the central C[triple bond]C triple bond were computed and correlated to each other. The linear dependence obtained for the two push-pull parameters d(C[triple bond]C) and pi*(C[triple bond]C)/pi(C[triple bond]C) quantifies changes in the push-pull effect of substituents while deviations from the best line of fit indicate and ascertain quantitatively to what extend the inductive (+/-I) substituent effect changes with respect to the bond length of the C[triple bond]C triple bond.