Cu,Zn-superoxide dismutase without Zn is folded but catalytically inactive.

Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)-Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.

Incorporating a completely renormalized coupled cluster approach into a composite method for thermodynamic properties and reaction paths.

The correlation consistent composite approach (ccCA), using the S4 complete basis set two-point extrapolation scheme (ccCA-S4), has been modified to incorporate the left-eigenstate completely renormalized coupled cluster method, including singles, doubles, and non-iterative triples (CR-CC(2,3)) as the highest level component. The new ccCA-CC(2,3) method predicts thermodynamic properties with an accuracy that is similar to that of the original ccCA-S4 method. At the same time, the inclusion of the single-reference CR-CC(2,3) approach provides a ccCA scheme that can correctly treat reaction pathways that contain certain classes of multi-reference species such as diradicals, which would normally need to be treated by more computationally demanding multi-reference methods. The new ccCA-CC(2,3) method produces a mean absolute deviation of 1.7 kcal/mol for predicted heats of formation at 298 K, based on calibration with the G2/97 set of 148 molecules, which is comparable to that of 1.0 kcal/mol obtained using the ccCA-S4 method, while significantly improving the performance of the ccCA-S4 approach in calculations involving more demanding radical and diradical species. Both the ccCA-CC(2,3) and ccCA-S4 composite methods are used to characterize the conrotatory and disrotatory isomerization pathways of bicyclo[1.1.0]butane to trans-1,3-butadiene, for which conventional coupled cluster methods, such as the CCSD(T) approach used in the ccCA-S4 model and, in consequence, the ccCA-S4 method itself might fail by incorrectly placing the disrotatory pathway below the conrotatory one. The ccCA-CC(2,3) scheme provides correct pathway ordering while providing an accurate description of the activation and reaction energies characterizing the lowest-energy conrotatory pathway. The ccCA-CC(2,3) method is thus a viable method for the analyses of reaction mechanisms that have significant multi-reference character, and presents a generally less computationally intensive alternative to true multi-reference methods, with computer costs and ease of use that are similar to those that characterize the more established, CCSD(T)-based, ccCA-S4 methodology.

Effect of spacer attachment sites and pH-sensitive headgroup expansion on cationic lipid-mediated gene delivery of three novel myristoyl derivatives.

The transfection activity and physicochemical properties of the dimyristoyl derivatives from three novel series of double-chained tertiary cationic lipids were compared. Two of the derivatives were constructed as isomers with different linkages of the same bis-(2-dimethylaminoethane) polar headgroup and hydrophobic chains to the diaminopropanol backbone, while the third was designed with a hydrophilic region containing only a single ionizable amine group. Such systematic molecular changes offer a great opportunity to delineate factors critical for transfection activity, which in this work include the intramolecular distance between the hydrophobic chains and pH-expandability of the polar headgroup. The physical studies comprised a variety of techniques, including pKa determination, Langmuir monolayer studies, fluorescence anisotropy, gel electrophoresis mobility shift assay, ethidium bromide displacement assay, particle size distribution, and zeta potential. These studies are crucial in the development of lipid-based gene delivery systems with improved efficacy. Physicochemical characterization revealed that a symmetric bivalent pH-expandable polar headgroup in combination with greater intramolecular space between the hydrophobic chains provide for high transfection activity through efficient binding and compaction of pDNA, increased acyl chain fluidity, and high molecular elasticity.