Specificity in transmembrane helix-helix interactions defines a hierarchy of stability for sequence variants,

The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alaninescanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several ''hot spots'' for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design. G enome sequencing efforts reveal that approximately 20% of ORFs in complex organisms may encode proteins containing at least one helical transmembrane segment (1). Despite these numbers, as well as the fact that membrane proteins carry out many essential cell functions, our understanding of the sequence-structure-function relationships for this class of proteins lags far behind that of soluble proteins. These realities underscore the importance of biophysical and structural work aimed toward understanding chemical principles of helical membrane protein structural stability. Because the phospholipid bilayer places structural constraints on a helical membrane protein, the folding of a polypeptide sequence into a helical membrane protein can be considered, experimentally and theoretically, in separable thermodynamic steps (2, 3). The usefulness of this framework arises from the fact that individual energetic processes can be independently studied. The principal features of a polypeptide sequence that will give rise to the formation of an independently stable transmembrane ␣-helix are generally known (3). This information has been used extensively in computational search algorithms with reasonable accuracy rates to identify potential helical transmembrane proteins (reviewed in ref. 3). Once this is accomplished, however, the helical membrane protein folding problem then becomes focused on understanding and predicting the side-to-side associations in which these preformed transmembrane ␣-helices will participate. It is this final thermodynamic step in helical membrane protein folding that we investigate in this study. In a continuing effort to understand the structural and energetic principles of the side-to-side interactions of transmembrane ␣-helices, we have quantitatively examined the sequence dependence of the glycophorin A transmembrane helix dimerization. The propensity of the glycophorin A transmembrane domain to dimerize in a sequence-specific manner has been a paradigm for study of transmembrane helix-helix association in hydrophobic environments (4-9). An additional advantage for detailed thermodynamic analysis of the GpA transmembrane segment (TMS) dimerization is the fact that a solution NMR structure has been solved (10). Together with considerations of principles of stability of helices in membranes, the NMR structure provides a three-dimensional model for interpretation of potential structural consequences due to mutation. Understanding the chemical principles driving the selfassociation of the glycophorin transmembrane ␣-helix is of particular interest because both the NMR structure and the exquisite sequence dependence determined by SDS͞PAGE suggest that a detailed geometry of van der Waals interactions specify and stabilize the dimer (4, 8, 10). Only one residue with a polar side-chain, Thr 87 , is found at the dimer interface. The solution NMR structure of the glycophorin A transmembrane dimer in dodecylphosphocholine micelles reveals no interhelical hydrogen bond at this position (10), although the recent solid state NMR data from Smith and coworkers (11) hints that Thr 87 might participate in an intermonomer hydrogen bond in lipid bilayers. Nevertheless, the energetic stabilization from such a hydrogen bond is uncertain. Recent studies on the introduction of polar side chains into model transmembrane peptides find that residues containing two polar side-chain atoms (such as asparagine) have a much greater tendency to drive transmembrane helix association than residues containing only one polar sidechain atom (threonine or serine; refs. 12 and 13). It has been proposed that side-chain rotamer entropy is not expected to play a large role in the self-association of the glycophorin A transmembrane ␣-helix (10). The interacting surface of the glycophorin A TMS contains only three residues with some rotamer freedom in an ␣-helix (Leu Abbreviations: TMS, transmembrane segment; C8E5, pentaoxylethylene-octylether. † To whom reprint requests should be addressed

The GxxxGcontaining transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions.

ABSTRACT: The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein-protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN-TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix-helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein-protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix-helix interactions. Receptor tyrosine kinases (RTKs) 1 are known to play an important role in the development and/or progression of many forms of cancer. A molecular understanding of the mechanisms by which these receptors promote cellular transformation is critical for the understanding and treatment of human cancer. In normal cells, RTKs are transmembrane signaling proteins that transmit biological signals from the extracellular environment to the interior of the cell. The regulated transmission of these signals is important for many cellular events, such as cell growth and differentiation, axonal growth, epithelial growth and development (1). RTKs are misregulated in many human cancers including breast cancer (erbB2/Her2) (2, 3), ovarian cancer (erbB2) (2, 3), melanoma (CCK4) (4), and colon cancer (erbB1 and CCK4) (5, 6). The predominant RTK-related genetic alteration in human cancer is not mutation of the receptor protein, but rather it is amplification of the gene. Subsequent overexpression of receptor proteins leads to constitutive stimulation of the RTK activity and uncontrolled cellular signaling. Recently, colon carcinoma kinase 4 (CCK4), a new member of the receptor tyrosine kinase superfamily was cloned from human colon carcinoma-derived cell lines (7). The CCK4 mRNA for this gene was variably expressed in colon carcinoma derived cell lines but not expressed in human adult colon tissues. In contrast, it is expressed in fetal colon of the mouse (7). These observations suggest that the normal role for CCK4 may be in development of the colon followed by down regulation in the adult tissue. The unusual expression of CCK4 in colon carcinoma cell lines might indicate that the protein is functioning abnormally in adult colon cells, which suggests a role in colon carcinoma development and/or proliferation. Orthologues for CCK4 have been cloned from other species (8-10). Amino acid residues within the transmembrane domain (TMD) are among the conserved features (9). The CCK4 TMD is 55% identical to the transmembrane sequences of the chicken Klg and Hydra Lemon orthologues (9), and a pattern of residues with helical periodicity is conserved. Within the conserved residues of the TMD is a GxxxG sequence, a motif thought to promote interactions between transmembrane helices * To whom correspondence should be addressed. Voice: 410-516-7256. Fax: 410-516-4118. E-mail: [email protected] 1 Abbreviations: CCK4, colon carcinoma kinase 4; C14 betaine, 3-(N,N-dimethylmyristyl-ammonio)propanesulfonate; RTK, receptor tyrosine kinases; SN, Staphylococcal nuclease; TMD, transmembrane domain; GpA, glycophorin A; SN-CCK4, a fusion protein composed of SN and the transmembrane domain of CCK4; SN-GpA, a fusion protein composed of SN and the transmembrane domain of glycophorin A

Changes in work habits of lifeguards in relation to Florida red tide

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Harmful Algae 9 (2010): 419-425, doi:10.1016/j.hal.2010.02.005.The marine dinoflagellate, Karenia brevis, is responsible for Florida red tides. Brevetoxins, the neurotoxins produced by K. brevis blooms, can cause fish kills, contaminate shellfish, and lead to respiratory illness in humans. Although several studies have assessed different economic impacts from Florida red tide blooms, no studies to date have considered the impact on beach lifeguard work performance. Sarasota County experiences frequent Florida red tides and staffs lifeguards at its beaches 365 days a year. This study examined lifeguard attendance records during the time periods of March 1 to September 30 in 2004 (no bloom) and March 1 to September 30 in 2005 (bloom). The lifeguard attendance data demonstrated statistically significant absenteeism during a Florida red tide bloom. The potential economic costs resulting from red tide blooms were comprised of both lifeguard absenteeism and presenteeism. Our estimate of the costs of absenteeism due to the 2005 red tide in Sarasota County is about $3,000. On average, the capitalized costs of lifeguard absenteeism in Sarasota County may be on the order of$100,000 at Sarasota County beaches alone. When surveyed, lifeguards reported not only that they experienced adverse health effects of exposure to Florida red tide but also that their attentiveness and abilities to take preventative actions decrease when they worked during a bloom, implying presenteeism effects. The costs of presenteeism, which imply increased risks to beachgoers, arguably could exceed those of absenteeism by an order of magnitude. Due to the lack of data, however, we are unable to provide credible estimates of the costs of presenteeism or the potential increased risks to bathers.This research was supported by the National Science Foundation under The Research Experience for Undergraduate Program, grant number 0453955; the P01 ES 10594, DHHS NIH of the National Institute of Environmental Health Sciences; the Center for Oceans and Human Health at the Woods Hole Oceanographic Institution [National Science Foundation (NSF) OCE-0430724; National Institute of Environmental Health Sciences (NIEHS) P50 ES012742]; and the Ocean and Human Health Center at the University of Miami Rosenstiel School (NSF 0CE0432368; NIEHS 1 P50 ES12736)

Structure of the lethal phage pinhole

Perhaps the simplest of biological timing systems, bacteriophage holins accumulate during the phage morphogenesis period and then trigger to permeabilize the cytoplasmic membrane with lethal holes; thus, terminating the infection cycle. Canonical holins form very large holes that allow nonspecific release of fully-folded proteins, but a recently discovered class of holins, the pinholins, make much smaller holes, or pinholes, that serve only to depolarize the membrane. Here, we interrogate the structure of the prototype pinholin by negative-stain transmission electron-microscopy, cysteine-accessibility, and chemical cross-linking, as well as by computational approaches. Together, the results suggest that the pinholin forms symmetric heptameric structures with the hydrophilic surface of one transmembrane domain lining the surface of a central channel ≈15 Å in diameter. The structural model also suggests a rationale for the prehole state of the pinholin, the persistence of which defines the duration of the viral latent period, and for the sensitivity of the holin timing system to the energized state of the membrane

Human responses to Florida red tides : policy awareness and adherence to local fertilizer ordinances

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Science of The Total Environment 493 (2014): 898-909, doi:10.1016/j.scitotenv.2014.06.083.To mitigate the damages of natural hazards, policy responses can be beneficial only if they are effective. Using a self-administered survey approach, this paper focuses on the adherence to local fertilizer ordinances (i.e., county or municipal rules regulating the application of fertilizer to private lawns or facilities such as golf courses) implemented in jurisdictions along the southwest Florida coast in response to hazardous blooms of Florida red tides (Karenia brevis). These ordinances play a role in the context of evolving programs of water pollution control at federal, state, water basin, and local levels. With respect to policy effectiveness, while the strength of physical linkages is of critical importance, the extent to which humans affected are aware of and adhere to the relevant rules, is equally critical. We sought to understand the public’s depth of understanding about the rationales for local fertilizer ordinances. Respondents in Sarasota, Florida, were asked about their fertilizer practices in an area that has experienced several major blooms of Florida red tides over the past two decades. A highly educated, older population of 305 residents and “snowbirds” reported relatively little knowledge about a local fertilizer ordinance, its purpose, or whether it would change the frequency, size, or duration of red tides. This finding held true even among subpopulations that were expected to have more interest in or to be more knowledgeable about harmful algal blooms. In the face of uncertain science and environmental outcomes, and with individual motivations at odds with evolving public policies, the effectiveness of local community efforts to decrease the impacts of red tides may be compromised. Targeted social-science research on human perceptions about the risks of Florida red tides and education about the rationales for potential policy responses is warranted.This work was funded under sponsorship of the National Science Foundation (NSF), awards #1009106 and #1004181and the National Institute for Environmental Health Sciences (NIEHS), award # R21ES017413-01A2. Fleming received support from the European Regional Development Fund and European Social Fund (European Centre for Environment and Human Health, University of Exeter Medical School)

The Allen Telescope Array Pi GHz Sky Survey I. Survey Description and Static Catalog Results for the Bootes Field

The Pi GHz Sky Survey (PiGSS) is a key project of the Allen Telescope Array. PiGSS is a 3.1 GHz survey of radio continuum emission in the extragalactic sky with an emphasis on synoptic observations that measure the static and time-variable properties of the sky. During the 2.5-year campaign, PiGSS will twice observe ~250,000 radio sources in the 10,000 deg^2 region of the sky with b > 30 deg to an rms sensitivity of ~1 mJy. Additionally, sub-regions of the sky will be observed multiple times to characterize variability on time scales of days to years. We present here observations of a 10 deg^2 region in the Bootes constellation overlapping the NOAO Deep Wide Field Survey field. The PiGSS image was constructed from 75 daily observations distributed over a 4-month period and has an rms flux density between 200 and 250 microJy. This represents a deeper image by a factor of 4 to 8 than we will achieve over the entire 10,000 deg^2. We provide flux densities, source sizes, and spectral indices for the 425 sources detected in the image. We identify ~100$ new flat spectrum radio sources; we project that when completed PiGSS will identify 10^4 flat spectrum sources. We identify one source that is a possible transient radio source. This survey provides new limits on faint radio transients and variables with characteristic durations of months.Comment: Accepted for publication in ApJ; revision submitted with extraneous figure remove

The employee as 'Dish of the Day’:human resource management and the ethics of consumption

This article examines the ethical implications of the growing integration of consumption into the heart of the employment relationship. Human resource management (HRM) practices increasingly draw upon the values and practices of consumption, constructing employees as the ‘consumers’ of ‘cafeteria-style’ benefits and development opportunities. However, at the same time employees are expected to market themselves as items to be consumed on a corporate menu. In relation to this simultaneous position of consumer/consumed, the employee is expected to actively engage in the commodification of themselves, performing an appropriate organizational identity as a necessary part of being a successful employee. This article argues that the relationship between HRM and the simultaneously consuming/consumed employee affects the conditions of possibility for ethical relations within organizational life. It is argued that the underlying ‘ethos’ for the integration of consumption values into HRM practices encourages a self-reflecting, self-absorbed subject, drawing upon a narrow view of individualised autonomy and choice. Referring to Levinas’ perspective that the primary ethical relation is that of responsibility and openness to the Other, it is concluded that these HRM practices affect the possibility for ethical being

The Allen Telescope Array Pi GHz Sky Survey I. Survey Description and Static Catalog Results for the Bootes Field

The Pi GHz Sky Survey (PiGSS) is a key project of the Allen Telescope Array. PiGSS is a 3.1 GHz survey of radio continuum emission in the extragalactic sky with an emphasis on synoptic observations that measure the static and time-variable properties of the sky. During the 2.5-year campaign, PiGSS will twice observe ~250,000 radio sources in the 10,000 deg^2 region of the sky with b > 30 deg to an rms sensitivity of ~1 mJy. Additionally, sub-regions of the sky will be observed multiple times to characterize variability on time scales of days to years. We present here observations of a 10 deg^2 region in the Bootes constellation overlapping the NOAO Deep Wide Field Survey field. The PiGSS image was constructed from 75 daily observations distributed over a 4-month period and has an rms flux density between 200 and 250 microJy. This represents a deeper image by a factor of 4 to 8 than we will achieve over the entire 10,000 deg^2. We provide flux densities, source sizes, and spectral indices for the 425 sources detected in the image. We identify ~100$ new flat spectrum radio sources; we project that when completed PiGSS will identify 10^4 flat spectrum sources. We identify one source that is a possible transient radio source. This survey provides new limits on faint radio transients and variables with characteristic durations of months.Comment: Accepted for publication in ApJ; revision submitted with extraneous figure remove