Proteins are Not Recruited: A Plea for Better Diction

Nearly all biological processes proceed or are controlled by protein-protein or protein-ligand binding reactions. Using anthropomorphic language to describe these interactions conveys an incorrect physical description of these processes while simultaneously minimizing the importance of the thermodynamics underpinning the associated interactions. Indeed, we should never say that proteins are recruited to binding partners or binding sites since this implies both a non- existent level of communication within biological systems and a non-existent process by which proteins or binding sites actively seek other proteins. Both of these fictions hinder our ability to determine quantitatively or qualitatively distinct biophysical descriptions of the associated systems. Here we present examples of how interactions typically described as protein recruitment can be more accurately and often more simply described as variations within binding equilibria. We argue that this approach is better for describing protein-protein and protein-ligand binding, even when the objective is only a qualitative description, especially for discussions with students in courses and research groups as it provides testable models for these interaction

Ab initio Berechnungen zur Anregung von Elektronen-Loch-Paaren durch Molekülschwingungen am Beispiel von HCl auf Al(111)

In dieser Dissertation wird zeitabhängige Dichtefunktionaltheorie mit Ehrenfestdynamik verwendet, um den Energietransfer eines vibrationsangeregtem Chlorwasserstoffmoleküls bei der Streuung an einer Aluminium-Oberfläche zu untersuchen. Die Berechnung und Untersuchung des elektronischen Grundzustands mittels Dichtefunktionaltheorie zeigt bereits die für den Energietransfer entscheidenden Eigenschaften des Moleküls und seiner Interaktion mit der Oberfläche. Die zeitabhängigen Rechnungen ergeben für das stark vibrationsangeregte Molekül große elektronisch nicht-adiabatische Effekte. Bei größerem Oberflächen-Molekül-Abstand kommt es zu einem stärkeren Energietransfer in das elektronische System. Es entsteht ein Maximum des Effekts bei einem Oberflächen-Molekül-Abstand, der größer ist als der Physisorptionsabstand des Moleküls. Unterschreitet die Vibrationsanregung eine von der Orientierung des Moleküls abhängige Grenze, so sind die nicht-adiabatischen Effekte nahezu Null

A Framework for Studying Crucial Steps in Proteasome Core Particle Assembly

The ability of proteins to repeatedly and reliably self-assemble in the cell is a critical element of the maintenance of life. Despite this, the mechanisms that underlie these events are poorly understood. The proteasome core particle from Rhodococcus erythropolis is an excellent model system for understanding assembly processes. This system has a two step assembly pathway where individual subunits first assemble into half proteasomes. Then, two half proteasomes dimerize to produce a full proteasome core particle. The beta subunit of this complex is synthesized in an inactive with an N-terminal propeptide that is cleaved after assembly is complete, rendering the CP enzymatically active. Evidence suggests that the propeptides plays a crucial role in both steps of the assembly process. To date, however, it has been impossible to fully characterize the role of the propeptide in assembly because this protein is typically produced as a heterogeneous mixture with a variety of N-terminally truncations in the propeptides itself. Here, we used Ligation Independent Cloning to produce a beta variant, which we call D3, that is homogeneous for the full-length propeptide. We also used Native PAGE to begin to characterize the kinetics of half proteasome dimerization. We found that there is a temperature-dependent effect on the dimerization process and that the presence of the full propeptide dramatically slowed assembly when compared to the heterogeneous beta. Using these methods, we can now study the thermodynamics and kinetics of this system much more rigorously than has been possible to date

Matter-wave interference and deflection of tripeptides decorated with fluorinated alkyl chains

Studies of neutral biomolecules in the gas phase allow for the study of molecular properties in the absence of solvent and charge effects, thus complementing spectroscopic and analytical methods in solution or in ion traps. Some properties, such as the static electronic susceptibility, are best accessed in experiments that act on the motion of the neutral molecules in an electric field. Here, we screen seven peptides for their thermal stability and electron impact ionizability. We identify two tripeptides as sufficiently volatile and thermostable to be evaporated and interfered in the long-baseline universal matter-wave interferometer. Monitoring the deflection of the interferometric molecular nanopattern in a tailored external electric field allows us to measure the static molecular susceptibility of Ala-Trp-Ala and Ala-Ala-Trp bearing fluorinated alkyl chains at C- and N-termini. The respective values are; 4; π; ε; 0; ×; 330; ±; 150; Å; 3; and; 4; π; ε; 0; ×; 270; ±; 80; Å; 3;

Mimicking Elementary Reactions of Manganese Lipoxygenase Using Mn-hydroxo and Mn-alkylperoxo Complexes

Manganese lipoxygenase (MnLOX) is an enzyme that converts polyunsaturated fatty acids to alkyl hydroperoxides. In proposed mechanisms for this enzyme, the transfer of a hydrogen atom from a substrate C-H bond to an active-site MnIII-hydroxo center initiates substrate oxidation. In some proposed mechanisms, the active-site MnIII-hydroxo complex is regenerated by the reaction of a MnIII-alkylperoxo intermediate with water by a ligand substitution reaction. In a recent study, we described a pair of MnIII-hydroxo and MnIII-alkylperoxo complexes supported by the same amide-containing pentadentate ligand (6Medpaq). In this present work, we describe the reaction of the MnIII-hydroxo unit in C-H and O-H bond oxidation processes, thus mimicking one of the elementary reactions of the MnLOX enzyme. An analysis of kinetic data shows that the MnIII-hydroxo complex [MnIII(OH)(6Medpaq)]+ oxidizes TEMPOH (2,2′-6,6′-tetramethylpiperidine-1-ol) faster than the majority of previously reported MnIII-hydroxo complexes. Using a combination of cyclic voltammetry and electronic structure computations, we demonstrate that the weak MnIII-N(pyridine) bonds lead to a higher MnIII/II reduction potential, increasing the driving force for substrate oxidation reactions and accounting for the faster reaction rate. In addition, we demonstrate that the MnIII-alkylperoxo complex [MnIII(OOtBu)(6Medpaq)]+ reacts with water to obtain the corresponding MnIII-hydroxo species, thus mimicking the ligand substitution step proposed for MnLOX

Genetic variation of TLR4 influences immunoendocrine stress response: an observational study in cardiac surgical patients

Introduction: Systemic inflammation (e.g. following surgery) involves Toll-like receptor (TLR) signaling and leads to an endocrine stress response. This study aims to investigate a possible influence of TLR2 and TLR4 single nucleotide polymorphisms (SNPs) on perioperative adrenocorticotropic hormone (ACTH) and cortisol regulation in serum of cardiac surgical patients. To investigate the link to systemic inflammation in this context, we additionally measured 10 different cytokines in the serum. Methods: 338 patients admitted for elective cardiac surgery were included in this prospective observational clinical cohort study. Genomic DNA of patients was screened for TLR2 and TLR4 SNPs. Serum concentrations of ACTH, cortisol, interferon (IFN)-, interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)- and granulocyte macro-phage-colony stimulating factor (GM-CSF) were determined before surgery, immediately post surgery and on the first postoperative day. Results: 13 patients were identified as TLR2 SNP carrier, 51 as TLR4 SNP carrier and 274 pa-tients as non-carrier. Basal levels of ACTH, cortisol and cytokines did not differ between groups. In all three groups a significant, transient perioperative rise of cortisol could be ob-served. However, only in the non-carrier group this was accompanied by a significant ACTH rise, TLR4 SNP carriers had significant lower ACTH levels compared to non-carriers ((mean[95% confidence intervals]) non-carriers: 201.9[187.7 to 216.1]pg/ml; TLR4 SNP car-riers: 149.9[118.4 to 181.5]pg/ml; TLR2 SNP carriers: 176.4[110.5 to 242.3]pg/ml). Compared to non-carriers, TLR4 SNP carriers showed significant lower serum IL-8, IL-10 and GM-CSF peaks ((mean[95% confidence intervals]): IL-8: non-carriers: 42.6[36.7 to 48.5]pg/ml, TLR4 SNP carriers: 23.7[10.7 to 36.8]pg/ml; IL-10: non-carriers: 83.8[70.3 to 97.4]pg/ml, TLR4 SNP carriers: 54.2[24.1 to 84.2]pg/ml; GM-CSF: non-carriers: 33.0[27.8 to 38.3]pg/ml, TLR4 SNP carriers: 20.2[8.6 to 31.8]pg/ml). No significant changes over time or between the groups were found for the other cytokines. Conclusions: Regulation of the immunoendocrine stress response during systemic inflamma-tion is influenced by the presence of a TLR4 SNP. Cardiac surgical patients carrying this ge-notype showed decreased serum concentrations of ACTH, IL-8, IL-10 and GM-CSF. This finding might have impact on interpreting previous and designing future trials on diagnosing and modulating immunoendocrine dysregulation (e.g. adrenal insufficiency) during systemic inflammation and sepsis

Electron-hole pairs during the adsorption dynamics of O2 on Pd(100) - Exciting or not?

During the exothermic adsorption of molecules at solid surfaces dissipation of the released energy occurs via the excitation of electronic and phononic degrees of freedom. For metallic substrates the role of the nonadiabatic electronic excitation channel has been controversially discussed, as the absence of a band gap could favour an easy coupling to a manifold of electronhole pairs of arbitrarily low energies. We analyse this situation for the highly exothermic showcase system of molecular oxygen dissociating at Pd(100), using time-dependent perturbation theory applied to first-principles electronic-structure calculations. For a range of different trajectories of impinging O2 molecules we compute largely varying electron-hole pair spectra, which underlines the necessity to consider the high-dimensionality of the surface dynamical process when assessing the total energy loss into this dissipation channel. Despite the high Pd density of states at the Fermi level, the concomitant non-adiabatic energy losses nevertheless never exceed about 5% of the available chemisorption energy. While this supports an electronically adiabatic description of the predominant heat dissipation into the phononic system, we critically discuss the non-adiabatic excitations in the context of the O2 spin transition during the dissociation process.Comment: 20 pages including 7 figures; related publications can be found at http://www.fhi-berlin.mpg.de/th/th.html [added two references, changed V_{fsa} to V_{6D}, modified a few formulations in interpretation of spin asymmetry of eh-spectra, added missing equals sign in Eg.(2.10)

Characterization of a transport activity for long-chain peptides in barley mesophyll vacuoles

The plant vacuole is the largest compartment in a fully expanded plant cell. While only very limited metabolic activity can be observed within the vacuole, the majority of the hydrolytic activities, including proteolytic activities reside in this organelle. Since it is assumed that protein degradation by the proteasome results in the production of peptides with a size of 3-30 amino acids, we were interested to show whether the tonoplast exhibits a transport activity, which could deliver these peptides into the vacuole for final degradation. It is shown here that isolated barley mesophyll vacuoles take up peptides of 9-27 amino acids in a strictly ATP-dependent manner. Uptake is inhibited by vanadate, but not by NH4+, while GTP could partially substitute for ATP. The apparent affinity for the 9 amino acid peptide was 15 μM, suggesting that peptides are efficiently transferred to the vacuole in vivo. Inhibition experiments showed that peptides with a chain length below 10 amino acids did not compete as efficiently as longer peptides for the uptake of the 9 amino acid peptide. Our results suggest that vacuoles contain at least one peptide transporter that belongs to the ABC-type transporters, which efficiently exports long-chain peptides from the cytosol into the vacuole for final degradatio

AtPTR4 and AtPTR6 are differentially expressed, tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectivel