Allosteric Activation of the Par-6 PDZ Via a Partial Unfolding Transition

Proteins exist in a delicate balance between the native and unfolded states, where thermodynamic stability may be sacrificed to attain the flexibility required for efficient catalysis, binding, or allosteric control. Partition-defective 6 (Par-6) regulates the Par polarity complex by transmitting a GTPase signal through the Cdc42/Rac interaction binding PSD-95/Dlg/ZO-1 (CRIB-PDZ) module that alters PDZ ligand binding. Allosteric activation of the PDZ is achieved by local rearrangement of the L164 and K165 side chains to stabilize the interdomain CRIB:PDZ interface and reposition a conserved element of the ligand binding pocket. However, microsecond to millisecond dynamics measurements revealed that L164/K165 exchange requires a larger rearrangement than expected. The margin of thermodynamic stability for the PDZ domain is modest (∼3 kcal/mol) and further reduced by transient interactions with the disordered CRIB domain. Measurements of local structural stability revealed that tertiary contacts within the PDZ are disrupted by a partial unfolding transition that enables interconversion of the L/K switch. The unexpected participation of partial PDZ unfolding in the allosteric mechanism of Par-6 suggests that native-state unfolding may be essential for the function of other marginally stable proteins

Quitters referring smokers: a quitline chain-referral pilot study

BACKGROUND: Telephone counseling Quitlines can support smoking cessation, but are under-utilized. We explored the use of smoker peer-referrals to increase use of a Quitline in Mississippi and Alabama. FINDINGS: Collaborating with the Alabama and Mississippi Quitline, we piloted peer-referrals to Quitlines. Successful \u27quitters\u27 who had used the Quitline were contacted at routine follow-up and recruited to participate as a peer-referrer and refer their friends and family who smoked to the Quitline. Peer-referrers completed a training session, received a manual and a set of Quitline brochures a peer-referral forms. These peer-referral forms were then returned to the Quitline telephone counselors who proactively called the referred smokers. Of the initial potential pool of 96 who quit using the Quitline, 24 peer-referrers (75% Women, 29% African-American, and high school graduates/GED 67%) were recruited and initially agreed to participate as peer-referrers. Eleven of the 24 who initially agreed were trained, and of these 11, 4 (4%) actively referred 23 friends and family over 2 months. From these 23 new referrals, three intakes (100% Women, 66% African-American) were completed. Of the initial pool of 96, 4 (4%) actively participated in referring friends and family. Quitline staff and peer-referrers noted several barriers including: time-point in which potential peer-referrers were asked to participate, an \u27overwhelming\u27 referral form to use and limited ways to refer. CONCLUSIONS: Though \u27quitters\u27 were willing to agree to peer-refer, we received a minority of referrals. However, we identified several areas to improve this new method for increasing awareness and access to support systems like the Quitline for smokers who want to quit

Genetic evidence that the Makira region in northeastern Madagascar is a hotspot of malaria transmission

Additional file 3: Figure S1. Contains the bootstrap re-sample statistics for comparing genetic relatedness

Health Information Seeking on Behalf of Others: Characteristics of ‘Surrogate Seekers’

Understanding the health information seeking behaviors of surrogate seekers (those who seek health information for others) may guide efforts to reach disadvantaged populations. We used 2011-2012 data from the Health Information National Trends Survey to describe the behaviors of online surrogate seekers. Respondents were asked about their use of the Internet for surrogate seeking over the prior 12 months. Data were weighted to calculate population estimates. Compared to those who sought health information online for only themselves, surrogate seekers were more likely to live in households with others (weighted percent 89.4% vs. 82.5% of self-seekers; p \u3c 0.05); no significant differences in gender, race, income or education were observed. Surrogate seekers were more likely to report: visits to social networking sites to read and share about medical topics; participation in online health support groups and downloading of health information to electronic devices. On multivariate analysis, those who had looked online for a healthcare provider were more likely to be surrogate seekers (OR 1.67, 95% CI 1.08-2.59). Our results offer insight for leveraging health communication efforts to reach populations who rely upon surrogate seekers for health information

Culturing of the first 37:4 predominant lacustrine haptophyte : geochemical, biochemical, and genetic implications

Author Posting. © The Author(s), 2011. 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 Geochimica et Cosmochimica Acta 78 (2012): 51–64, doi:10.1016/j.gca.2011.11.024.Long chain alkenones (LCAs) are potential biomarkers for quantitative paleotemperature reconstructions from lacustrine environments. However, progress in this area has been severely hindered by the lack of culture studies of haptophytes responsible for alkenone distributions in lake sediments: the predominance of C37:4 LCA. Here we report the first enrichment culturing of a novel haptophyte phylotype (Hap-A) from Lake George, ND that produces predominantly C37:4-LCA. Hap-A was enriched from its resting phase collected from deep sediments rather than from water column samples. In contrast, enrichments from near surface water yielded a different haptophyte phylotype (Hap-B), closely related to Chrysotila lamellosa and Pseudoisochrysis paradoxa, which does not display C37:4-LCA predominance (similar enrichments have been reported previously). The LCA profile in sediments resembles that of Hap-A enrichments, suggesting that Hap-A is the dominant alkenone producer of the sedimentary LCAs. In enrichments, excess lighting appeared to be crucial for triggering blooms of Hap-A. Both and indices show a linear relationship with temperature for Hap-A in enrichments, but the relationship appears to be dependent on the growth stage. Based on 18S rRNA gene analyses, several lakes from the Northern Great Plains, as well as Pyramid Lake, NV and Tso Ur, Tibetan Plateau, China contain the same two haptophyte phylotypes. The Great Plains lakes show the Hap-A-type LCA distribution, whereas Pyramid and Tso Ur show the Hap-B type distribution. Waters of the Great Plain lakes are dominated by sulfate, whereas those Pyramid and Tso Ur are dominated by carbonate, suggesting that the sulfate to carbonate ratio may be a determining factor for the competitiveness of the Hap-A and Hap-B phylotypes in natural settings.This work was supported by a grant from the National Science Foundation to Y. Huang (EAR06-02325) and a Brown University Graduate School Dissertation Fellowship to J. L. Toney

Rapid sulfurisation of highly branched isoprenoid (HBI) alkenes in sulfidic Holocene sediments from Ellis Fjord, Antarctica

Author Posting. © Elsevier B.V., 2007. 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 Organic Geochemistry 38 (2007): 128-139, doi:10.1016/j.orggeochem.2006.08.003.Samples of particulate organic matter from the water column and anoxic Holocene sediment layers from the Small Meromictic Basin (SMB) in Ellis Fjord (eastern Antarctica) were analyzed to study the early incorporation of reduced inorganic sulfur species into highly branched isoprenoid (HBI) alkenes. HBIs were not detected in the water column samples from austral winter, whereas compounds containing the C25 HBI skeleton were abundant in all analyzed Holocene sediment layers. The structure of the C25:2 HBI alkene together with its enriched stable carbon isotopic composition suggest that the HBI alkene is produced by a diatom or diatoms probably belonging to the Navicula genus present in the sea-ice which covers the area most of the year. Within just 500 years of deposition, all of the HBI alkene was sulfurised. A mixture of products was formed, including components tentatively identified as a C25 HBI thiane and three S-containing dimers composed of two C25:1 HBI skeletons linked together by a sulfide bond. Most of the HBI alkene, however, was converted to polar S-containing compounds. The observed reaction rate for sulfurisation the C25:2 HBI alkene is the highest observed so far in natural systems. Sterols and other lipids known to be prone to sulfurisation were only minimally sulfurised under these depositional conditions. The reason for this is presently unclear.Funding for the collection of the sediment and water samples (by MJLC and CW) was provided by ASAC grant 1166 to JKV. This work was further supported by a grant from the Netherlands Organization for Scientific Research (NWO; Netherlands Antarctic Research Proposals 851.20.006 to JSSD)

An NMR structural study of nickel-substituted rubredoxin

J Biol Inorg Chem (2010) 15:409–420 DOI 10.1007/s00775-009-0613-6The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The βCH2 proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine Hβ protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH–S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin

Resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Protein Science (2002), 11:2464–2470Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S]center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with 15N or 15N + 13C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D 15N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the 13C shifts were obtained from an HNCA experiment. Comparison of 1H–15N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues 8.5 Å from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits

Switchable Membrane Remodeling and Antifungal Defense by Metamorphic Chemokine XCL1

Antimicrobial peptides (AMPs) are a class of molecules which generally kill pathogens via preferential cell membrane disruption. Chemokines are a family of signaling proteins that direct immune cell migration and share a conserved α–β tertiary structure. Recently, it was found that a subset of chemokines can also function as AMPs, including CCL20, CXCL4, and XCL1. It is therefore surprising that machine learning based analysis predicts that CCL20 and CXCL4’s α-helices are membrane disruptive, while XCL1’s helix is not. XCL1, however, is the only chemokine known to be a metamorphic protein which can interconvert reversibly between two distinct native structures (a β-sheet dimer and the α–β chemokine structure). Here, we investigate XCL1’s antimicrobial mechanism of action with a focus on the role of metamorphic folding. We demonstrate that XCL1 is a molecular “Swiss army knife” that can refold into different structures for distinct context-dependent functions: whereas the α–β chemokine structure controls cell migration by binding to G-Protein Coupled Receptors (GPCRs), we find using small angle X-ray scattering (SAXS) that only the β-sheet and unfolded XCL1 structures can induce negative Gaussian curvature (NGC) in membranes, the type of curvature topologically required for membrane permeation. Moreover, the membrane remodeling activity of XCL1’s β-sheet structure is strongly dependent on membrane composition: XCL1 selectively remodels bacterial model membranes but not mammalian model membranes. Interestingly, XCL1 also permeates fungal model membranes and exhibits anti-Candida activity in vitro, in contrast to the usual mode of antifungal defense which requires Th17 mediated cell-based responses. These observations suggest that metamorphic XCL1 is capable of a versatile multimodal form of antimicrobial defense

Narratives to enhance smoking cessation interventions among African-American smokers, the ACCE project

BACKGROUND: Low-income, African-American smokers are less likely to have resources to aid in quitting smoking. Narrative communication may provide an enhancement to traditional smoking cessation interventions like NRT, medications, or behavioral treatments for this audience. After extensive pilot testing of stories and personal experiences with smoking cessation from African-Americans from a low-income community, we conducted a randomized control trial using stories to augment routine inpatient treatment among African-Americans at an urban Southern hospital (N = 300). RESULTS: Differences in smoking cessation outcomes between the intervention (stories DVD + routine clinical treatment) and control (routine clinical treatment) arms were compared using self-report and carbon monoxide measurement at 6-months. Compared to control, individuals who viewed the intervention stories DVD reported greater intentions to quit. Although continuous quitting marginally favored the intervention, our main result did not reach statistical significance (p = 0.16). CONCLUSION: Narrative communication via storytelling to promote smoking cessation among African-Americans in the South is one method to communicate smoking cessation. Results suggest this may not be sufficient as a stand-alone augmentation of routine clinical treatment for continuous smoking cessation. Smoking cessation efforts need to continually assess different means of communicating to smokers about quitting. CLINICAL TRIALS REGISTRATION: The ClinicalTrials.gov Identifier is NCT00101491. This trial was registered January 10, 2005