Femtosecond observation of benzyne intermediates in a molecular beam: Bergman rearrangement in the isolated molecule

In this communication, we report our femtosecond real-time observation of the dynamics for the three didehydrobenzene molecules (p-, m-, and o-benzyne) generated from 1,4-, 1,3-, and 1,2-dibromobenzene, respectively, in a molecular beam, by using femtosecond time-resolved mass spectrometry. The time required for the first and the second C-Br bond breakage is less than 100 fs; the benzyne molecules are produced within 100 fs and then decay with a lifetime of 400 ps or more. Density functional theory and high-level ab initio calculations are also reported herein to elucidate the energetics along the reaction path. We discuss the dynamics and possible reaction mechanisms for the disappearance of benzyne intermediates. Our effort focuses on the isolated molecule dynamics of the three isomers on the femtosecond time scale.

Dynamical hierarchy in transition states: Why and how does a system climb over the mountain?

How a reacting system climbs through a transition state during the course of a reaction has been an intriguing subject for decades. Here we present and quantify a technique to identify and characterize local invariances about the transition state of an N-particle Hamiltonian system, using Lie canonical perturbation theory combined with microcanonical molecular dynamics simulation. We show that at least three distinct energy regimes of dynamical behavior occur in the region of the transition state, distinguished by the extent of their local dynamical invariance and regularity. Isomerization of a six-atom Lennard–Jones cluster illustrates this: up to energies high enough to make the system manifestly chaotic, approximate invariants of motion associated with a reaction coordinate in phase space imply a many-body dividing hypersurface in phase space that is free of recrossings even in a sea of chaos. The method makes it possible to visualize the stable and unstable invariant manifolds leading to and from the transition state, i.e., the reaction path in phase space, and how this regularity turns to chaos with increasing total energy of the system. This, in turn, illuminates a new type of phase space bottleneck in the region of a transition state that emerges as the total energy and mode coupling increase, which keeps a reacting system increasingly trapped in that region.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.