In adaptation biology the discovery of intracellular osmolyte molecules that in some cases reach molar levels, raises questions of how they influence protein thermodynamics. We’ve addressed such questions using the premise that from atomic coordinates, the transfer free energy of a native protein (ΔGtr,N) can be predicted by summing measured water-to-osmolyte transfer free energies of the protein’s solvent exposed side chain and backbone component parts. ΔGtr,D is predicted using a self avoiding random coil model for the protein, and ΔGtr,D − ΔGtr,N, predicts the m-value, a quantity that measures the osmolyte effect on the N ⇌ D transition. Using literature and newly measured m-values we show 1:1 correspondence between predicted and measured m-values covering a range of 12 kcal/mol/M in protein stability for 46 proteins and 9 different osmolytes. Osmolytes present a range of side chain and backbone effects on N and D solubility and protein stability key to their biological roles.

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Telomeres are specialized chromatin structures that protect chromosome ends from nucleolytic processing by DNA repair machinery. The foundation of human telomere structure consists of a long array of tandem duplex DNA sequences (TTAGGG) and terminates with a single-stranded 3' end. To protect the chromosome end, telomeres are thought to adopt a lariat structure known as a telomere-loop (T-loop)1. T-loops are stabilized by DNA displacement loops (D-loops) generated by the invasion of a single-stranded telomeric DNA tail into an adjacent region of duplex telomere. Recent studies suggest that telomere-associated proteins promote strand invasion during telomeric D-loop formation through the application of torque to the DNA2. Although the molecular mechanism of T-loop formation has been described using biochemical approaches, the torque response and internal structural equilibrium of duplex telomeric DNA are not well characterized. To probe the mechanical properties of duplex telomeric DNA, we developed a magnetic tweezers assay to detect the response of single telomeric DNA molecules to precisely applied degrees of tension and torque. Rotation-extension curves under varying tension demonstrate that the repetitive telomere DNA sequence is more refractory to torque-induced denaturation than a non-telomeric control molecule of comparable GC content. In addition, force-extension analysis of negatively supercoiled telomeric DNA in the presence of different counter-ions (K þ vs. Li þ ), reveals that transient torque-induced denaturation of duplex telomeric DNA promotes a structural transition into stable DNA G-quadruplexes. Lastly, using a single molecule DNA topology-based assay, we directly monitor the torquedependent invasion of single stranded telomere DNA primers into duplex telomeric DNA tethers. Our results provide insight into the molecular mechanisms of telomere-associated proteins and enzymes during structural remodeling of telomeres. Small molecules (osmolytes) are known to either stabilize or destabilize proteins/nucleotides depending on the concentrations and/or solvent conditions. The presence of different molecules and ions in the surrounding medium affects the stability of DNA in solution. In this work, we have developed a High-Throughput method for quantifying the energetic impact of addition of various osmolytes on short DNA duplexes. Six 19-base pair, non-self-complementary duplex DNA oligomers along with a 16-base pair control duplex DNA, having varied GC-content (ranging from 16% to 79%), nearest neighbors and end sequences were used. We sampled thirteen different osmolytes that are common in humans and throughout nature by covering different chemical classes including, sugars, polyols, amino acids, and methylamines. Varying concentrations of these osmolytes (from 0.5 M up to 3.0 M) were examined for their effects on these duplexes. Experiments were performed in 384-well plates that were prepared using a robotic device, which was calibrated for the correct dispense volume for different components of th...