VB/MM protein landscapes: a study of the S(N)2 reaction in haloalkane dehalogenase.

QM/MM methods are widely used for studies of reaction mechanisms in water and protein environments. Recently, we have developed the VB/MM method in which the QM part is implemented by the ab initio valence bond (VB) method. Here, we report on further improvement of the VB/MM method which makes it possible to use the method for reactivity studies in systems where the QM and MM parts are connected by covalent bonds followed by first ab initio VB study of reactivity in proteins. We implemented a simple link atom scheme to treat the boundary interactions. We tested the performance of the link atom treatment in combination with the VB/MM method on an S(N)2 reaction and found it to be sufficiently accurate. We then used the VB/MM method to study the S(N)2 reaction in haloalkane dehalogenase (DhlA). We show that the predicted reaction barrier heights are in good agreement with estimated experimental values, thereby validating the method. Finally, we analyze the reaction energetics in terms of contributions of the VB configurations and conclude that such analysis is instrumental in pinpointing the essential features of the catalytic mechanism.

What is the role of the helical domain of Gsalpha in the GTPase reaction?

Structural analysis of Gsalpha shows that it is composed of two domains: the ras-like domain (RD) that is conserved in all members of the GTPase superfamily and is homologous to the monomeric G-proteins (e.g., p21ras) and an alpha-helical domain (HD) that is unique to heterotrimeric G-proteins. Little is known about the function of the HD. Recent experiments by Bourne and co-workers, who expressed both the RD and the HD of Gsalpha separately and found that GTP hydrolysis is very slow if only recombinant RD is present but is accelerated when the HD is added, suggest that the HD serves as an intrinsic GTPase-activating protein (GAP). In this work, the GTP hydrolysis in Gsalpha was studied. The results obtained by calculating catalytic effects with and without the HD provide evidence for the role of the HD as a GAP. It is demonstrated that a major part of the catalysis is obtained because of an allosteric influence of the HD on the RD. Structural as well as energetic considerations suggest that the HD confines the RD to a more compact conformation, pushing the phosphate into an orientation where it is further stabilized, thus lowering the overall reaction barrier. The resemblance between the behavior of rasGAP and the HD suggests that the conclusion may be a general conclusion, applicable for all of the G-protein members.

Dynamical singularities for complex initial conditions and the motion at a real separatrix.

This work investigates singularities occurring at finite real times in the classical dynamics of one-dimensional double-well systems with complex initial conditions. The objective is to understand the relationship between these singularities and the behavior of the systems for real initial conditions. An analytical treatment establishes that the dynamics of a quartic double well system possesses a doubly infinite sequence of singularities. These are associated with initial conditions that converge to those for the real separatrix as the singularity time becomes infinite. This confluence of singularities is shown to lead to the unstable behavior that characterizes the real motion at the separatrix. Numerical calculations confirm the existence of a large number of singularities converging to the separatrix for this and two additional double-well systems. The approach of singularities to the real axis is of particular interest since such behavior has been related to the formation of chaos in nonintegrable systems. The properties of the singular trajectories which cause this convergence to the separatrix are identified. The hyperbolic fixed point corresponding to the potential energy maximum, responsible for the characteristic motion at a separatrix, also plays a critical role in the formation of the complex singularities by delaying trajectories and then deflecting them into asymptotic regions of space from where they are directly repelled to infinity in a finite time.