Deuteration of nonexchangeable protons on proteins affects their thermal stability, side-chain dynamics, and hydrophobicity.

We have investigated the effect of deuteration of non-exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well-known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G-binding domain of protein G (GB3). Reversed-phase high-performance liquid chromatography (RP-HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees. RP-HPLC also permitted quantification of the effect of deuteration of just three core phenylalanine side chains of HP36. NMR dynamics investigation has focused on methyl axes motions using cross-correlated relaxation measurements. The analysis of order parameters provided a complex picture indicating that deuteration generally increases motional amplitudes of sub-nanosecond motion in GB3 but decreases those in HP36. Combined with earlier dynamics measurements at Cα -Cß sites and backbone sites of GB3, which probed slower time scales, the results point to the need to probe multiple atoms in the protein and variety of time scales to the discern the full complexity of the effects of deuteration on dynamics.

Synthesis of Altrose Poly-amido-saccharides with ß-N-(1→2)-d-amide Linkages: A Right-Handed Helical Conformation Engineered in at the Monomer Level.

The design and synthesis of amide-linked saccharide oligomers and polymers, which are predisposed to fold into specific ordered secondary structures, is of significant interest. Herein, right-handed helical poly amido-saccharides (PASs) with ß-N-(1→2)-d-amide linkages are synthesized by the anionic ring-opening polymerization of an altrose ß-lactam monomer (alt-lactam). The right-handed helical conformation is engineered into the polymers by preinstalling the ß configuration of the lactam ring in the monomer via the stereospecific [2+2] cycloaddition of trichloroacetyl isocyanate with a d-glycal possessing a 3-benzyloxy group oriented to the α-face of the pyranose. The tert-butylacetyl chloride initiated polymerization of the alt-lactam proceeds smoothly to afford stereoregular polymers with narrow dispersities. Birch reduction of the benzylated polymers gives water-soluble altrose PASs (alt-PASs) in high yields without degradation of the polymer backbone. Circular dichroism analysis shows the alt-PASs adopt a right-handed helical conformation in aqueous solutions. This secondary conformation is stable over a wide range of different conditions, such as pH (2.0 to 12.0), temperature (5 to 75 °C), ionic salts (2.0 M LiCl, NaCl, and KCl), as well as in the presence of protein denaturants (4.0 M urea and guanidinium chloride). Cytotoxicity studies reveal that the alt-PASs are nontoxic to HEK, HeLa, and NIH3T3 cells. The results showcase the ability to direct solution conformation of polymers through monomer design. This approach is especially well-suited and straightforward for PASs as the helical conformations formed result from constraints imposed by the relatively rigid and sterically bulky repeating units.

Combined Molecular Dynamics Simulations and Experimental Studies of the Structure and Dynamics of Poly-Amido-Saccharides.

Poly-amido-saccharides (PAS) are carbohydrate-based, enantiopure synthetic polymers in which sugar repeat units are joined by amide linkages. This unique and relatively rigid pyranose backbone contributes to their defined helical secondary structure and remarkable chemical properties. Glucose- (glc-) and galactose- (gal-) PAS 10-mer structures are synthesized and investigated with molecular dynamics (MD) simulations and experimental measurements. Quantum mechanical DFT energy minimization calculations, as well as experimental observables including circular dichroism, (1)H,(13)C-HSQC, and (1)H,(1)H-NOESY 2D-NMR studies, validated the all-atom simulation models produced using a modified CHARMM force field. Water radial distribution functions show distinct differences in the glc- and gal-PAS systems that correlate well with observed differences in solubility between gal-PASs and glc-PASs. The computational analysis and MD simulations are in good agreement with experimental results, validating the proposed models as reliable representations of novel glc- and gal-PASs.