Two Snapshots of Electron Transport across the Membrane: INSIGHTS INTO THE STRUCTURE AND FUNCTION OF DsbD*S?

In Escherichia coli, the periplasmic protein disulfide isomerase, DsbC, is maintained reduced by transfer of electrons from cytoplasmic thioredoxin-1 (Trx1) via the cytoplasmic membrane protein, DsbD. The transmembrane domain of DsbD (DsbDβ), which comprises eight transmembrane segments (TMs), contains two redox-active cysteines (Cys-163 and Cys-285), each of which is water-exposed to both sides of the membrane. Cys-163 in TM1 and Cys-285 in TM4 can interact with cytoplasmic Trx1 and a periplasmic Trx-like domain of DsbD, respectively. When Cys-163 and Cys-285 are disulfide-bonded, the C-terminal halves of TM1 and TM4 are water-exposed, whereas the N-terminal halves of these TMs are not. To assess possible conformational changes of DsbDβ when its two cysteines are reduced, we have determined the accessibility of portions of TM1 and TM4. We substituted cysteines for amino acids in these TM segments and determined alkylation accessibility. We find that the alkylation accessibility of single Cys replacements in TM1 and TM4 is the same in oxidized and reduced DsbDβ, indicating a relatively static conformation of DsbDβ between the two redox states. We also find that the accessibility of amino acids of TM2 and TM3 when Cys-163 and Cys-285 are oxidized or reduced shows no change. Together, these results support a relatively static structure of DsbDβ in the switch between the oxidized and the reduced state but raise the possibility of conformational changes when interacting with Trx proteins. In addition, we also find water-exposed residues in the cytoplasmic proximal portion of TM3, allowing a more detailed characterization of the cavity in DsbDβ.The cell envelope of most bacteria is an oxidizing environment. In many bacteria, the main oxidant system consists of DsbA and DsbB. DsbA introduces disulfide bonds into newly synthesized and secreted polypeptides containing cysteines and is regenerated as an oxidative enzyme by the membrane protein DsbB. Electrons are ultimately transferred from DsbB to the respiratory chain (1–3). However, there are also certain cell envelope proteins that require a reductive enzyme to act on them. This is the case for those proteins that contain multiple cysteines and that are often misoxidized by DsbA, thus generating non-native disulfide bonds. The protein DsbC, a protein disulfide isomerase, can promote rearrangement of such incorrect disulfide bonds, resulting in a correctly folded protein (4–7). It does this either by using the reduced cysteine in its active site to resolve non-native disulfide bonds and promoting the formation of the native pairs or simply by reducing the substrate protein, which may be correctly oxidized by DsbA and given a second chance (8). In the latter mechanism, DsbC becomes oxidized and must be reduced. This reduction is carried out by a cytoplasmic membrane protein, DsbD, which receives electrons for this purpose from thioredoxin-1 (Trx1)² in the cytoplasm (5, 9).DsbD is composed of three domains, each containing two redox-active cysteines (Fig. 1). DsbDβ, the membrane-embedded domain containing eight transmembrane segments (TMs), receives electrons from Trx1 and then transfers them to the C-terminal periplasmic domain, DsbDγ, which contains a Trx-like fold (10–15). The N-terminal periplasmic domain, DsbDα, which contains an immunoglobulin-like fold, is then reduced by DsbDγ and transfers electrons to DsbC (13, 16, 17).Open in a separate windowFIGURE 1.Electron transfer pathway through transmembrane domain (β) of DsbD and its membrane topology predicted from the primary sequence. The topology of DsbDβ was predicted using HMMTOP. The essential two cysteines are shown in bold without a circle and numbered. The residues indicated with a star in TM1 and TM4 are water-exposed when the Cys-163 and Cys-285 are disulfide-bonded (19). Studies on the residues in TM2 and TM3 are shown in Fig. 4. The essential cysteines in the other domains (α and γ) and interacting proteins (Trx1 and DsbC) are shown in a white S (the sulfur of thiol) in gray circles. The tailless arrows indicate where a signal sequence of DsbD and three hemagglutinin (HA) epitopes are fused at the N terminus of DsbDβ, and a c-Myc epitope is fused at the C terminus of DsbDβ. The figure in the inset describes the model of DsbDβ, in which Cys-163 and Cys-285 form a disulfide bond in the middle of the protein and halves of C-terminal TM1 and TM4 are water-exposed (black; C-TM1 and C-TM4), whereas those of N-terminal ones are not (N-TM1 and N-TM4).Electron transfer through the transmembrane domain DsbDβ is quite unusual when compared with that of other membrane electron transport proteins. Required extrinsic factors, for example, quinone, FAD, heme, or metal centers, which are often used as cofactors for electron transfer, have not been found (18). As a result, it is proposed that thiol-disulfide exchange reactions alone promote the transfer of electrons across the cytoplasmic membrane, utilizing the two cysteines, Cys-163 and Cys-285 of DsbDβ. Evidence for this mechanism comes from the detection of likely reaction intermediates including the Cys-163–Cys-285 disulfide and mixed disulfide complexes, Trx1-DsbDβ(Cys-163) and DsbDβ(Cys-285)-DsbDγ (13, 14, 19).We have previously studied the accessibility to the aqueous environment of amino acids in TM1 and TM4 of DsbDβ, which contain Cys-163 and Cys-285, respectively. Our results, in conjunction with a comparison of the amino acid sequences of TM1–3 and TM4–6 of DsbDβ, suggest antiparallel and pseudosymmetrical properties of TM1 and TM4 (19, 20). Cys-163 in TM1 and Cys-285 in TM4 are water-exposed to both sides of the membrane when they are in the reduced state and suggested to be located in the middle of the membrane (helices). When Cys-163 and Cys-285 are disulfide-bonded, the proximal portion of the cytoplasmic side of TM1 at the C terminus of Cys-163 and that of the periplasmic side of TM4 at the C terminus of Cys-285 are highly water-exposed, whereas the other portion of each TM is not. Therefore, we proposed an hourglass-like model (Fig. 1, inset) and suggested that the water-exposed halves of TM1 and TM4 are cavity-located non-membrane-spanning helices and involved in the interactions of Trx1 and DsbDγ, respectively.However, in our previous studies (19), we did not determine whether, when DsbDβ is in the reduced state, the arrangement of TM1 and TM4 or other TMs would be similar to that seen when Cys-163 and Cys-285 are disulfide-bonded. Doing this comparison is important because it has been proposed that alternative exposure of the cysteines to the aqueous environment depending on their redox states explains the electron transfer process across the membrane (Refs. 21–23; see “Discussion”). In addition, to further define the structural features of DsbDβ, we wished to determine how TM segments other than TM1 and TM4 are arranged in terms of their water accessibility. In this study, we examined the accessibility of many residues in TM1 and TM4, as well as studying the arrangements of TM2 and TM3, using site-directed cysteine alkylation in both redox states.Our data show that the four TMs studied, TM1, TM2, TM3, and TM4, have similar accessibility properties whether DsbDβ is in the oxidized or reduced state. We also find additional water-exposed residues in the proximal portion of the cytoplasmic side of TM3.     

Head impact exposure in collegiate football players

In American football, impacts to the helmet and the resulting head accelerations are the primary cause of concussion injury and potentially chronic brain injury. The purpose of this study was to quantify exposures to impacts to the head (frequency, location and magnitude) for individual collegiate football players and to investigate differences in head impact exposure by player position. A total of 314 players were enrolled at three institutions and 286,636 head impacts were recorded over three seasons. The 95th percentile peak linear and rotational acceleration and HITsp (a composite severity measure) were 62.7g, 4378rad/s(2) and 32.6, respectively. These exposure measures as well as the frequency of impacts varied significantly by player position and by helmet impact location. Running backs (RB) and quarter backs (QB) received the greatest magnitude head impacts, while defensive line (DL), offensive line (OL) and line backers (LB) received the most frequent head impacts (more than twice as many than any other position). Impacts to the top of the helmet had the lowest peak rotational acceleration (2387rad/s(2)), but the greatest peak linear acceleration (72.4g), and were the least frequent of all locations (13.7%) among all positions. OL and QB had the highest (49.2%) and the lowest (23.7%) frequency, respectively, of front impacts. QB received the greatest magnitude (70.8g and 5428rad/s(2)) and the most frequent (44% and 38.9%) impacts to the back of the helmet. This study quantified head impact exposure in collegiate football, providing data that is critical to advancing the understanding of the biomechanics of concussive injuries and sub-concussive head impacts.     

Head impact exposure in male and female collegiate ice hockey players

The purpose of this study was to quantify head impact exposure (frequency, location and magnitude of head impacts) for individual male and female collegiate ice hockey players and to investigate differences in exposure by sex, player position, session type, and team. Ninety-nine (41 male, 58 female) players were enrolled and 37,411 impacts were recorded over three seasons. Frequency of impacts varied significantly by sex (males: 287 per season, females: 170, p<0.001) and helmet impact location (p<0.001), but not by player position (p=0.088). Head impact frequency also varied by session type; both male and female players sustained more impacts in games than in practices (p<0.001), however the magnitude of impacts did not differ between session types. There was no difference in 95th percentile peak linear acceleration between sexes (males: 41.6 g, females: 40.8 g), but 95th percentile peak rotational acceleration and HITsp (a composite severity measure) were greater for males than females (4424, 3409 rad/s², and 25.6, 22.3, respectively). Impacts to the back of the helmet resulted in the greatest 95th percentile peak linear accelerations for males (45.2 g) and females (50.4 g), while impacts to the side and back of the head were associated with the greatest 95th percentile peak rotational accelerations (males: 4719, 4256 rad/sec², females: 3567, 3784 rad/sec² respectively). It has been proposed that reducing an individual's head impact exposure is a practical approach for reducing the risk of brain injuries. Strategies to decrease an individual athlete's exposure need to be sport and gender specific, with considerations for team and session type.     

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.     

The effects of structure-conformation modifications of melanotropin analogs on learning and memory: D-amino acid substituted linear and cyclic analogs

Alpha-MSH has a wide variety of putative biological activities in addition to its classical melanocyte dispersing activity. Since each of these activities appears to be mediated by a discrete receptor, this peptide is an excellent candidate for exploring conformational restrictions which determine the chemical-physical basis for hormone action on specific activities. Experiments One and Two evaluated several cyclic and linear analogs of alpha-MSH on retrieval of memory during the reactivation of memory for a passive avoidance response following hypothermia-induced amnesia. Three of the cyclic analogs appear to have enhanced the peptide's ability to serve as a reactivation agent. One of the linear Nle4,D-Phe7 analogs antagonized whereas three others enhanced reactivation. The D-Phe7 substitution in cyclic analogs did not affect reactivation. Another group of animals were trained on a step-through passive avoidance task and tested 25 days later. The cyclic analog enhanced memory whereas the D-Phe7 analog and alpha-MSH had no effect. Finally, two analogs were tested on a black-white discrimination. Although the cyclic analog had no effect on either acquisition or reversal of this learning, the Nle4,D-Phe7 analog significantly impaired reversal learning. The results from these preliminary studies suggest that structural modifications of alpha-MSH do alter its potency and pattern of actions in learning and memory situations.     

Evidence for specificity at an early step in protein export in Escherichia coli.

We previously described mutations in a gene, secB, which have pleiotropic effects on protein export in Escherichia coli. In this paper, we report the isolation of mutants in which the activity of the secB gene was eliminated. Null mutations in secB affected only a subset of exported proteins. Strains carrying these mutations, although unable to grow on L broth plates, were still viable on minimal media. These secB mutations reversed a block in the translation of an exported protein that was caused by the elimination of another component of the secretion machinery, SecA protein. These results suggest that the secB product acts at an early step in the export process and is involved in the export of only a subset of cell envelope proteins.     

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding

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Gut bacterial assemblages of freshwater macroinvertebrate functional feeding groups

The use of remote sensing for monitoring of submerged aquatic vegetation (SAV) in fluvial environments has been limited by the spatial and spectral resolution of available image data. The absorption of light in water also complicates the use of common image analysis methods. This paper presents the results of a study that uses very high-resolution image data, collected with a Near Infrared sensitive DSLR camera, to map the distribution of SAV species for three sites along the Desselse Nete, a lowland river in Flanders, Belgium. Plant species, including Ranunculus peltatus, Callitriche obtusangula, Potamogeton natans L., Sparganium emersum R. and Potamogeton crispus L., were classified from the data using object-based image analysis and expert knowledge. A classification rule set based on a combination of both spectral and structural image variation (e.g. texture and shape) was developed for images from two sites. A comparison of the classifications with manually delineated ground truth maps resulted for both sites in 61% overall accuracy. Application of the rule set to a third validation image resulted in 53% overall accuracy. These consistent results not only show promise for species-level mapping in such biodiverse environments but also prompt a discussion on assessment of classification accuracy.     

Localization of the Escherichia coli cell division protein FtsI (PBP3) to the division site and cell pole

FtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli . FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.