Expression systems for bovine rhodopsin: a review of the progress made in the Khorana laboratory

Here I will review the development of gene expression systems for production of bovine rhodopsin in the Khorana laboratory with particular focus on stable mammalian cell lines made using human embryonic kidney cells (HEK293S). The synthesis of a gene encoding bovine rhodopsin was completed in 1986. This gene was expertly designed with the built-in capacity for DNA duplex cassette replacement mutagenesis which made site-directed mutagenesis relatively straightforward. Intense effort was expended over several years in order to identify a gene expression system capable of producing rhodopsin in milligram amounts as required for biophysical studies. Mammalian expression systems, both transient and stable, were found to be the most favourable based on several criteria including receptor expression levels, correct folding and post translational processing, and capacity for purification of fully functional receptor. Transient expression using COS-1 cells was preferred for routine small-scale production of rhodopsin mutants, while HEK293S stable cell lines were used when milligram amounts of rhodopsin mutants were needed; for example, when conducting NMR studies

Structure and functional motifs of GCR1, the only plant protein with a GPCR fold?

Whether GPCRs exist in plants is a fundamental biological question. Interest in deorphanizing new G protein coupled receptors (GPCRs), arises because of their importance in signaling. Within plants, this is controversial as genome analysis has identified 56 putative GPCRs, including GCR1 which is reportedly a remote homologue to class A, B and E GPCRs. Of these, GCR2, is not a GPCR; more recently it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix alignment method, which has been benchmarked against the the class A – class B - class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologues to class A, class B and class F GPCRs, and shown that GCR1 is closer to class A and /or class B GPCRs than class A, class B or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the 6 GPCR classes. Variability comparisons provide additional evidence that GCR1 homologues have the GPCR fold. From the alignments and a GCR1 comparative model we have identified motifs that are common to GCR1, class A, B and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fol

Assessing the effect of dynamics on the closed-loop protein-folding hypothesis

The closed-loop (loop-n-lock) hypothesis of protein folding suggests that loops of about 25 residues, closed through interactions between the loop ends (locks), play an important role in protein structure. Coarse-grain elastic network simulations, and examination of loop lengths in a diverse set of proteins, each supports a bias towards loops of close to 25 residues in length between residues of high stability. Previous studies have established a correlation between total contact distance (TCD), a metric of sequence distances between contacting residues (cf. contact order), and the log-folding rate of a protein. In a set of 43 proteins, we identify an improved correlation ( r 2 = 0.76), when the metric is restricted to residues contacting the locks, compared to the equivalent result when all residues are considered ( r 2 = 0.65). This provides qualified support for the hypothesis, albeit with an increased emphasis upon the importance of a much larger set of residues surrounding the locks. Evidence of a similar-sized protein core/extended nucleus (with significant overlap) was obtained from TCD calculations in which residues were successively eliminated according to their hydrophobicity and connectivity, and from molecular dynamics simulations. Our results suggest that while folding is determined by a subset of residues that can be predicted by application of the closed-loop hypothesis, the original hypothesis is too simplistic; efficient protein folding is dependent on a considerably larger subset of residues than those involved in lock formation. </jats:p

Molecular genetic analysis of extracellular enzyme secretion by Erwinia carotovora

Erwinia carotovora subsp. carotovora (Ecc) secretes a variety of extracellular enzymes, namely pectinases (Pel), cellulases (Cel) and proteases (Prt). Some of these extracellular enzymes are considered to be the major pathogenicity determinants of this bacterium. Using the chemical mutagen ethyl methyl sulphonate (EMS), a range of Ecc mutants defective in extracellular enzyme production have been generated. One class was found to be pleiotropically defective in the production of Pel and Cel but unaffected for Prt production. Pel and Cel were still synthesised in this class of mutant but both enzymes accumulated within the periplasm. Mutants of the Pel-, Cel-, Prt+ class have been termed Out- mutants. A single Out- mutant, RJP190, was partially resistant to infection by two Ecc bacteriophages. Using a cosmid library of wild-type Ecc, 12 of the 14 Out- mutants were complemented to Out+. Further analysis of the complementing cosmids led to the identification of at least six out loci. A 3.7 kb region of DNA containing out genes was sequenced. This fragment of DNA overlapped with other out genes sequenced In this laboratory. The contiguous DNA (5.7 kb) encoded four proteins, OutD, OutE, OutF and OutG, which were visualised using a T7 directed expression system. The predicted Out proteins were found to share homology with other eubacterial proteins involved in macromolecular trafficking. Accumulated findings strongly suggest that this Out-type system is the major pathway used by Gram-negative bacteria for secreting proteins to the extracellular milieu

Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β2-adrenergic receptor

AbstractSequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix–helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2–H4 in the β2-adrenergic receptor (β2-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly3157.42 and Ser3197.46, on H7 for structure-function analysis. Replacing Ser3197.46 with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15–20% agonist-independent activity. Replacement of Ser3197.46 with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly3157.42 and Ser3197.46 are stabilizing β2-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly3157.42 with Trp2866.48 and hydrogen bonding interactions of Ser3197.46 with amino acids on H1–H2–H7 and with structural water

Short-range ordering in a battery electrode, the 'cation-disordered' rocksalt Li1.25Nb0.25Mn0.5O2.

Cation order, with a local structure related to γ-LiFeO2, is observed in the nominally cation-disordered Li-excess rocksalt Li1.25Nb0.25Mn0.5O2via X-ray diffraction, neutron pair distribution function analysis, magnetic susceptibility and NMR spectroscopy. The correlation length of ordering depends on synthesis conditions and has implications for the electrochemistry of these phases.EPSRC: EP/L015978/1 Basic Energy Science, US Department of Energy: DE-SC001258

Structural origins of voltage mysteresis in the Na-Ion cathode P2-Na0.67[Mg0.28Mn0.72]O2 : A combined spectroscopic and density functional theory study

Funding Information: E.N.B. acknowledges funding from the Engineering Physical Sciences Research Council (EPSRC) via the National Productivity Interest Fund (NPIF) 2018 and is also grateful for use of the ARCHER UK National Supercomputing Service via our membership in the UK’s HEC Materials Chemistry Consortium, funded by the EPSRC (EP/L000202). Research was also carried out at the Center for Functional Nanomaterials, Brookhaven National Laboratory, through the U.S. Department of Energy, Office of Basic Energy Sciences, Contract DE-AC02-98CH10866. P.J.R. thanks the Northeast Centre for Chemical Energy Storage (NECCES), an Energy Frontier Research Centre funded by the US Department of Energy, Office of Basic Energy Sciences, award DE-SC0012583. M.A.J. is grateful for the financial support of the EPSRC Centre for Doctoral Training (CDT) in Nanoscience and Nanotechnology Award EP/L015978/1. J.L. was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. 2019R1A6A1A10073437). E.N.B. also wishes to thank Dr M.F. Groh for assistance with setting up capillary XRD measurements. Publisher Copyright: ©Peer reviewedPublisher PD

Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation

The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff’s base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation

Hypersomnolence and Sleep-related Complaints in Metropolitan, Urban, and Rural Georgia

Persistent daytime hypersomnolence is associated with significant morbidity and mortality, but its prevalence in the population has been poorly documented. This study sought to characterize the prevalence of persistent daytime hypersomnolence, difficulties initiating and maintaining sleep, unrefreshing sleep, snoring, and the presence of physician-diagnosed sleep disorders in metropolitan, urban, and rural US Georgia populations. Between September 2004 and July 2005, a total of 6,530 randomly selected well and unwell adults, identified by screening interviews of 10,837 households (contacted by random digit dialing), completed a detailed phone interview. Sixteen percent reported persistent problems staying awake during the day; 26% reported persistent problems falling asleep at night; 31% experienced problems sleeping through the night; 34% were bothered by unrefreshing sleep; and 33% reported that they snored. In spite of the high occurrence of reported persistent sleep problems, only 10% of the survey participants reported having been diagnosed with a sleep disorder. These study findings highlight the need for increased public and clinician awareness with respect to proactively indentifying signs and symptoms of sleep disorders, a better understanding of their adverse impact upon morbidity and mortality, and their negative impact upon socioeconomic and academic potential

Estimating bycatch mortality for marine mammals : concepts and best practices

Support for this project was provided by the Lenfest Ocean Program (Contract ID: #31008).Fisheries bycatch is the greatest current source of human-caused deaths of marine mammals worldwide, with severe impacts on the health and viability of many populations. Recent regulations enacted in the United States under the Fish and Fish Product Import Provisions of its Marine Mammal Protection Act require nations with fisheries exporting fish and fish products to the United States (hereafter, “export fisheries”) to have or establish marine mammal protection standards that are comparable in effectiveness to the standards for United States commercial fisheries. In many cases, this will require estimating marine mammal bycatch in those fisheries. Bycatch estimation is conceptually straightforward but can be difficult in practice, especially if resources (funding) are limiting or for fisheries consisting of many, small vessels with geographically-dispersed landing sites. This paper describes best practices for estimating bycatch mortality, which is an important ingredient of bycatch assessment and mitigation. We discuss a general bycatch estimator and how to obtain its requisite bycatch-rate and fisheries-effort data. Scientific observer programs provide the most robust bycatch estimates and consequently are discussed at length, including characteristics such as study design, data collection, statistical analysis, and common sources of estimation bias. We also discuss alternative approaches and data types, such as those based on self-reporting and electronic vessel-monitoring systems. This guide is intended to be useful to managers and scientists in countries having or establishing programs aimed at managing marine mammal bycatch, especially those conducting first-time assessments of fisheries impacts on marine mammal populations.Publisher PDFPeer reviewe