Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long‐range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side‐chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side‐chain reorientation of R82 modulates the hydrogen‐bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton‐transfer in the methyl guanidinium‐hydronium‐hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O′ state where the proton on D85 is transferred to D212. Proteins 2016; 84:639–654. © 2016 Wiley Periodicals, Inc.     

A new direction for differentiating animal activity based on measuring angular velocity about the yaw axis

1. The use of animal‐attached data loggers to quantify animal movement has increased in popularity and application in recent years. High‐resolution tri‐axial acceleration and magnetometry measurements have been fundamental in elucidating fine‐scale animal movements, providing information on posture, traveling speed, energy expenditure, and associated behavioral patterns. Heading is a key variable obtained from the tandem use of magnetometers and accelerometers, although few field investigations have explored fine‐scale changes in heading to elucidate differences in animal activity (beyond the notable exceptions of dead‐reckoning).
2. This paper provides an overview of the value and use of animal heading and a prime derivative, angular velocity about the yaw axis, as an important element for assessing activity extent with potential to allude to behaviors, using “free‐ranging” Loggerhead turtles (Caretta caretta) as a model species.
3. We also demonstrate the value of yaw rotation for assessing activity extent, which varies over the time scales considered and show that various scales of body rotation, particularly rate of change of yaw, can help resolve differences between fine‐scale behavior‐specific movements. For example, oscillating yaw movements about a central point of the body's arc implies bouts of foraging, while unusual circling behavior, indicative of conspecific interactions, could be identified from complete revolutions of the longitudinal axis.
4. We believe this approach should help identification of behaviors and “space‐state” approaches to enhance our interpretation of behavior‐based movements, particularly in scenarios where acceleration metrics have limited value, such as for slow‐moving animals.

     

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.     

Computational analysis of photosynthetic systems

The use of various computational techniques for the study of photosynthetic systems is described ranging from genome analysis to density functional simulations of the oxygen evolving complex of PSII. The use of simulations for analyzing protein structures can aid in clarifying ambiguous and incomplete experimental results to identifying underlying rules to create efficient light-initiated charge separation at high efficiency.     

Dynamics of the DNA repair proteins WRN and BLM in the nucleoplasm and nucleoli

We have investigated the mobility of two EGFP-tagged DNA repair proteins, WRN and BLM. In particular, we focused on the dynamics in two locations, the nucleoli and the nucleoplasm. We found that both WRN and BLM use a “DNA-scanning” mechanism, with rapid binding–unbinding to DNA resulting in effective diffusion. In the nucleoplasm WRN and BLM have effective diffusion coefficients of 1.62 and 1.34 μm²/s, respectively. Likewise, the dynamics in the nucleoli are also best described by effective diffusion, but with diffusion coefficients a factor of ten lower than in the nucleoplasm. From this large reduction in diffusion coefficient we were able to classify WRN and BLM as DNA damage scanners. In addition to WRN and BLM we also classified other DNA damage proteins and found they all fall into one of two categories. Either they are scanners, similar to WRN and BLM, with very low diffusion coefficients, suggesting a scanning mechanism, or they are almost freely diffusing, suggesting that they interact with DNA only after initiation of a DNA damage response.     

The pKa Cooperative: a collaborative effort to advance structure-based calculations of pKa values and electrostatic effects in proteins

The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise.