The energetic contributions of hydrogen bonding to protein folding are still unclear, despite over 70 years of study. This is due partly to the difficulty of extracting thermodynamic information about specific interactions from protein mutagenesis data, and partly to the context dependence of hydrogen bond strengths. Herein, we test the hypothesis that hydrogen bond strengths depend on the polarity of their microenvironment, with stronger hydrogen bonds forming in non-polar surroundings. Double mutant thermodynamic cycle analysis using a combination of amide-to-ester backbone mutagenesis and traditional side chain mutagenesis revealed that hydrogen bonds can be stronger by up to 1.2 kcal mol−1 when they are sequestered in hydrophobic surroundings than when they are solvent exposed. Such large coupling energies between hydrogen bond strengths and local polarity suggest that the context dependence of hydrogen bond strengths must be accounted for in any comprehensive account of the forces responsible for protein folding.

We describe a new molecular class of genetic-pairing system that has a native DNA backbone but has all four base pairs replaced by new, larger pairs. The base pairs include size-expanded analogs of thymine and of adenine, both extended by the width of a benzene ring (2.4 A). The expanded-diameter double helices are more thermodynamically stable than the Watson-Crick helix, likely because of enhanced base stacking. Structural data confirm a right-handed, double-stranded, and base-paired helical form. Because of the larger base size, all the pairs of this helical system are fluorescent, which suggests practical applications in detection of natural DNA and RNA. Our findings establish that there is no apparent structural or thermodynamic prohibition against genetic systems having sizes different from the natural one.

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Facile labeling of proteins of interest is highly desirable in proteomic research as well as in the development of protein therapeutics.

Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+ -dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development.