Dimeric SecA Couples the Preprotein Translocation in an Asymmetric Manner

The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles

Preprotein mature domains contain translocase targeting signals that are essential for secretion

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion

A holin and an endopeptidase are essential for chitinolytic protein secretion in <i>Serratia marcescens</i>

Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria

Increasing the X-ray Diffraction Power of Protein Crystals by Dehydration: The Case of Bovine Serum Albumin and a Survey of Literature Data

Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA) free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA), presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 Å, b = 44.72 Å, c = 140.18 Å, β = 114.5°. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals

SecA-Mediated Protein Translocation through the SecYEG Channel

In bacteria, the Sec translocase mediates the translocation of proteins into and across the cytoplasmic membrane. It consists of a protein conducting channel SecYEG, the ATP-dependent motor SecA, and the accessory SecDF complex. Here we discuss the function and structure of the Sec translocase

Interference control in children with attention deficit/hyperactivity disorder

The view that Attention Deficit/Hyperactivity Disorder (ADHD) is associated with a diminished ability to control interfference is controversial and based exclusively on results of (verbal)-visual interference tasks, primarily the Stroop Color Word task. The present study compares medication-naïve children with ADHD (n∈=∈35 and n∈=∈51 in Experiments 1 and 2, respectively) with normal controls (n∈=∈26 and n∈=∈32, respectively) on two interference tasks to assess interference control in both the auditory and the visual modality: an Auditory Stroop task and a Simon task. Both groups showed reliable but equal degrees of interference on both tasks, suggesting that children with ADHD do not differ from normal controls in their ability to control interference in either modality. © 2008 The Author(s)

Inhibition, Reinforcement Sensitivity and Temporal Information Processing in ADHD and ADHD+ODD: Evidence of a Separate Entity?

This study compared children with ADHD-only, ADHD+ODD and normal controls (age 8–12) on three key neurocognitive functions: response inhibition, reinforcement sensitivity, and temporal information processing. The goal was twofold: (a) to investigate neurocognitive impairments in children with ADHD-only and children with ADHD+ODD, and (b) to test whether ADHD+ODD is a more severe from of ADHD in terms of neurocognitive performance. In Experiment 1, inhibition abilities were measured using the Stop Task. In Experiment 2, reinforcement sensitivity and temporal information processing abilities were measured using a Timing Task with both a reward and penalty condition. Compared to controls, children with ADHD-only demonstrated impaired inhibitory control, showed more time underestimations, and showed performance deterioration in the face of reward and penalty. Children with ADHD+ODD performed in-between children with ADHD-only and controls in terms of inhibitory controls and the tendency to underestimate time, but were more impaired than controls and children with ADHD-only in terms of timing variability. In the face of reward and penalty children with ADHD+ODD improved their performance compared to a neutral condition, in contrast to children with ADHD-only. In the face of reward, the performance improvement in the ADHD+ODD group was disproportionally larger than that of controls. Taken together the findings suggest that, in terms of neurocognitive functioning, comorbid ADHD+ODD is a substantial different entity than ADHD-only

Investigating Homology between Proteins using Energetic Profiles

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology