Peakmatch: a simple and robust method for peak list matching

Peak lists are commonly used in NMR as input data for various software tools such as automatic assignment and structure calculation programs. Inconsistencies of chemical shift referencing among different peak lists or between peak and chemical shift lists can cause severe problems during peak assignment. Here we present a simple and robust tool to achieve self-consistency of the chemical shift referencing among a set of peak lists. The Peakmatch algorithm matches a set of peak lists to a specified reference peak list, neither of which have to be assigned. The chemical shift referencing offset between two peak lists is determined by optimizing an assignment-free match score function using either a complete grid search or downhill simplex optimization. It is shown that peak lists from many different types of spectra can be matched reliably as long as they contain at least two corresponding dimensions. Using a simulated peak list, the Peakmatch algorithm can also be used to obtain the optimal agreement between a chemical shift list and experimental peak lists. Combining these features makes Peakmatch a useful tool that can be applied routinely before automatic assignment or structure calculation in order to obtain an optimized input data set.     

Automated solid-state NMR resonance assignment of protein microcrystals and amyloids

Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218–289) and α-synuclein yielded 88–97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77–90 % correctness if also assignments classified as tentative by the algorithm are included.     

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.     

Global analysis of phosphoproteome regulation by the Ser/Thr phosphatase Ppt1 in Saccharomyces cerevisiae

Even though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identify cellular protein targets of a serine/threonine phosphatase. We used SILAC-based quantitative MS to measure differences in protein expression and phosphorylation upon ablation of the serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide fractionation by strong cation exchange chromatography combined with immobilized metal affinity chromatography (IMAC) enrichment enabled quantification of more than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient yeast cells. We further quantified the relative expression of 1897 yeast proteins and detected no major protein changes accompanying Ppt1 deficiency. Notably, we found 33 phosphorylation sites to be significantly and reproducibly up-regulated while no phosphorylation events were repressed in cells lacking Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and site-specific fashion. Several of the regulated phosphoproteins were involved in the response to heat stress in agreement with known Ppt1 functions. Additionally, biosynthetic enzymes were particularly prominent among Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the control of various metabolic functions. These results demonstrate the utility of large-scale and quantitative phosphoproteomics to identify cellular sites of serine/threonine phosphatase action in an unbiased manner.     

Endosymbiosestudien an schildläusen. III. Macrocerococc us UND puto zwei primitive pseudococcinen

Ohne Zusammenfassung     

Hsp90 & Co. - a holding for folding.

Hsp90 is an abundant molecular chaperone that is involved in the folding of a defined set of signalling molecules including steroid-hormone receptors and kinases. Recent in vitro experiments suggest that Hsp90 contains two different binding sites for non-native proteins, which allow it to combine the properties of a promiscuous chaperone with those of a dedicated folding-helper protein. Significant progress has been made in analysing co-chaperones, which form defined, substrate-dependent complexes with Hsp90 in vivo. Structural studies have identified the ATP-binding site in the N-terminal domain of Hsp90, which can be blocked by high-affinity inhibitors. Although a detailed understanding of the mechanism of Hsp90 action is still lacking, recent advances suggest that the protein is the centre of a dynamic, multifunctional and multicomponent chaperone machinery that extends the limits of protein folding in the cell.     

Evaluating the Use of Global Sensitivity Analysis in Dynamic MFA

Dynamic material flow analysis (MFA) provides information about material usage over time and consequent changes in material stocks and flows. In order to understand the effect of limited data quality and model assumptions on MFA results, the use of sensitivity analysis methods in dynamic MFA studies has been on the increase. So far, sensitivity analysis in dynamic MFA has been conducted by means of a one‐at‐a‐time method, which tests parameter perturbations individually and observes the outcomes on output. In contrast to that, variance‐based global sensitivity analysis decomposes the variance of the model output into fractions caused by the uncertainty or variability of input parameters. The present study investigates interaction and time‐delay effects of uncertain parameters on the output of an archetypal input‐driven dynamic material flow model using variance‐based global sensitivity analysis. The results show that determining the main (first‐order) effects of parameter variations is often sufficient in dynamic MFA because substantial effects attributed to the simultaneous variation of several parameters (higher‐order effects) do not appear for classical setups of dynamic material flow models. For models with time‐varying parameters, time‐delay effects of parameter variation on model outputs need to be considered, potentially boosting the computational cost of global sensitivity analysis. Finally, the implications of exploring the sensitivities of model outputs with respect to parameter variations in the archetypical model are used to derive model‐ and goal‐specific recommendations on choosing appropriate sensitivity analysis methods in dynamic MFA.