Techniques for Small Groups

...I want to start out tonight by saying the two are very close together, Christ and the body of Christ, closer than you think. And renewal will come to you as you give your heart again to Christ himself. And as you give your heart again to the body of Christ, it is so important that you fall more and more in love with him

Protein evolution

On the origin of proteins A series of mistakes over the past 3.7 billion years or so has left us with a spectacular array of protein structures and functions, which are responsible for nothing less than life itself, writes Bea Perk

Theophany and Chaoskampf : the interpretation of Theophanic imagery in the Baal Epic, Isaiah, and the Twelve

This dissertation concerns the interpretation of theophanic imagery in the Prophets. In essence, my argument is that the imagery which attends and describes theophany in the poetic books of the Hebrew Bible is mythic and not metaphorical-that it should be interpreted in relation to the Chaoskampf myth, as Yahweh defeats chaos and restores order, rather than being understood as a metaphorical comparison with natural phenomena. In arguing this way, however, I am taking a new approach to theophanic imagery, for such imagery has almost uniformly been understood in Old Testament scholarship as metaphor. Before examining in detail this traditional and more widespread approach to theophanic imagery and the alternative to it which I wish to suggest, however, it will be helpful to state at the outset two foundations upon which my argument rests and the specific texts which will be enlisted to support it

Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become “stuck” in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations to trigger a profound evolutionary change in the protein's function and shaped the genetics of evolutionary reversibility.</p

The lncRNA Growth Arrest Specific 5 regulates cell survival via distinct structural modules with independent functions

There is increasing evidence that the architecture of long non-coding RNAs (lncRNAs) just like that of proteins-is hierarchically organized into independently folding sub-modules with distinct functions. Studies characterizing the cellular activities of such modules, however, are rare. The lncRNA growth arrest specific 5 (GAS5) is a key regulator of cell survival in response to stress and nutrient availability. We use SHAPE-MaP to probe the structure of GAS5 and identify three separate structural modules that act independently in leukemic T cells. The 5' terminal module with low secondary structure content affects basal survival and slows the cell cycle, whereas the highly structured core module mediates the effects of mammalian target of rapamycin (mTOR) inhibition on cell growth. These results highlight the central role of GAS5 in regulating cell survival and reveal how a single lncRNA transcript utilizes a modular structure-function relationship to respond to a variety of cellular stresses under various cellular conditions

Crystal Structure of an Ancient Protein: Evolution by Conformational Epistasis

The structural mechanisms by which proteins have evolved new functions are known only indirectly. We report x-ray crystal structures of a resurrected ancestral protein—the ∼450 million year-old precursor of vertebrate glucocorticoid (GR) and mineralocorticoid (MR) receptors. Using structural, phylogenetic, and functional analysis, we identify the specific set of historical mutations that recapitulate the evolution of GR’s hormone specificity from an MR-like ancestor. These substitutions repositioned crucial residues to create new receptor-ligand and intra-protein contacts. Strong epistatic interactions occur because one substitution changes the conformational position of another site. “Permissive” mutations—substitutions of no immediate consequence, which stabilize specific elements of the protein and allow it to tolerate subsequent function-switching changes—played a major role in determining GR’s evolutionary trajectory

Crystallization and preliminary X-ray diffraction analysis of Sfh3, a member of the Sec14 protein superfamily

Sec14 is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the yeast Saccharomyces cerevisiae and is the founding member of the Sec14 protein superfamily. Recent functional data suggest that Sec14 functions as a nanoreactor for PtdCho-regulated presentation of PtdIns to PtdIns kinase to affect membrane trafficking. Extrapolation of this concept to other members of the Sec14 superfamily suggests a mechanism by which a comprehensive cohort of Sec14-like nanoreactors sense correspondingly diverse pools of lipid metabolites. In turn, metabolic information is translated to signaling circuits driven by phosphoinositide metabolism. Sfh3, one of five Sec14 homologs in yeast, exhibits several interesting functional features, including its unique localization to lipid particles and microsomes. This localization forecasts novel regulatory interfaces between neutral lipid metabolism and phosphoinositide signaling. To launch a detailed structural and functional characterization of Sfh3, the recombinant protein was purified to homogeneity, diffraction-quality crystals were produced and a native X-ray data set was collected to 2.2 Å resolution. To aid in phasing, SAD X-ray diffraction data were collected to 1.93 Å resolution from an SeMet-labeled crystal at the Southeast Regional Collaborative Access Team at the Advanced Photon Source. Here, the cloning and purification of Sfh3 and the preliminary diffraction of Sfh3 crystals are reported, enabling structural analyses that are expected to reveal novel principles governing ligand binding and functional specificity for Sec14-superfamily proteins

Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1

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