Collagen is the most abundant protein in animals. This fibrous, structural protein comprises a righthanded bundle of three parallel, left-handed polyproline II-type helices. Much progress has been made in elucidating the structure of collagen triple helices and the physicochemical basis for their stability. New evidence demonstrates that stereoelectronic effects and preorganization play a key role in that stability. The fibrillar structure of type I collagen-the prototypical collagen fibril-has been revealed in detail. Artificial collagen fibrils that display some properties of natural collagen fibrils are now accessible using chemical synthesis and self-assembly. A rapidly emerging understanding of the mechanical and structural properties of native collagen fibrils will guide further development of artificial collagenous materials for biomedicine and nanotechnology.

Summary The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.

Cas9-based technologies have transformed genome engineering and the interrogation of genomic functions, but methods to control such technologies across numerous dimensions—including dose, time, specificity, and mutually exclusive modulation of multiple genes—are still lacking. We conferred such multidimensional controls to diverse Cas9 systems by leveraging small-molecule-regulated protein degron domains. Application of our strategy to both Cas9-mediated genome editing and transcriptional activities opens new avenues for systematic genome interrogation.

Steric and stereoelectronic effects play a defining role in molecular conformation and reactivity. In small molecules, steric and stereoelectronic effects often have dichotomous consequences. For example, the anomeric effect in glycosides yields axial substituents that are disfavored by sterics. 1 Similarly, replacing the steric effect of a methyl group with the stereoelectronic effect of a fluoro group enables a β-peptide to fold. 2 Stereoelectronic effects contribute markedly to the conformational stability of an abundant protein: collagen. 345 Collagen is a fibrous protein comprising a bundle of three parallel strands folded into polyproline type II helices. 6 Each strand consists of ∼300 repeats of the unit: XaaYaa-Gly, where Xaa is often (2S)-proline (Pro) and Yaa is often (2S,4R)-4-hydroxyproline (Hyp). The pyrrolidine ring in the Xaa and Yaa positions have C γ -endo and C γ -exo ring puckers, respectively. 7 These puckers can be preordained by a stereoelectronic effect. Specifically, the gauche effect from a 4S fluoro group stabilizes the C γ -endo pucker; that from a 4R fluoro group stabilizes the C γ -exo pucker (Figure 1). 3 These stereoelectronic effects can marked ly enhance the conformational stability of a collagen triple helix. We reasoned that pyrrolidine ring pucker could instead be fixed and hence collagen stability enhanced by steric rather than stereoelectronic effects. Herein, we report on the bestowal of conformational stability to collagen by steric effects that reiterate stereoelectronic effects.Density functional theory indicated that the pyrrolidine ring of (2S,4R)-4-methylproline (mep) has a strong preference (1.4 kcal/mol) for the C γ -endo pucker and that of (2S,4S)-4-methylproline (Mep) has a strong preference (1.7 kcal/mol) for the C γ -exo pucker ( Figure 1). These conformational preferences are observed in crystalline Ac-mep-NHMe and Ac-MepNHMe, 8 and follow the trend observed in 4-tert-butylprolines. 9 In the preferred conformations, the methyl group of mep and Mep adopts a pseudo-equatorial conformation. 10 A methyl group in this conformation should protrude radially from a collagen triple helix and thus not instill any deleterious interstrand steric interactions. Accordingly, we synthesized mepOH and MepOH 11 and incorporated these nonnatural amino acids into collagen strands to yield: (mep-Pro-Gly) 7 , (Pro-Mep-Gly) 7 , and (mep-Mep-Gly) 7 . We incubated solutions of each strand at ≤4 °C, and then used circular dichroism (CD) spectroscopy to detect formation of triple helices and assess their conformational stability.(mep-Pro-Gly) 7 , (Pro-Mep-Gly) 7 , and (mep-Mep-Gly) 7 formed triple helices at 4 °C, as indicated by an ellipiticity maximum near 225 nm (Figure 2A). The self-association of (ProMep-Gly) 7 , (mep-Mep-Gly) 7 , and, to a lesser extent, (mep-Pro-Gly) 7 at 4 °C was confirmed by sedimentation equilibrium experiments. 12 (mep-Pro-Gly) 7 , (Pro-Mep-Gly) 7 , and (mep- NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptMep-Gly) 7 triple helices ha...