Protein Factory Reveals Its Secrets (Part 4 End)

A similar proton-shuttle mechanism had been proposed earlier by professor of theoretical chemistry Johan Åqvist of Uppsala University, in Sweden, and coworkers. They based their proposal on molecular dynamics and combined quantum mechanical/molecular mechanics simulations. Similar simulations by chemistry professor Arieh Warshel and coworkers at the University of Southern California, Los Angeles, support the proton-shuttle mechanism as well, although the USC group found that electrostatic stabilization-not substrate assistance or orientational entropy-accounts for most of the catalytic effect.
Computation and simulation are likely to be extraordinarily useful for further clarifying the ribosome's mechanism, because these techniques currently represent "the only way to study the ribosome in motion in atomic detail," says theoretical biologist Kevin Sanbonmatsu of Los Alamos National Laboratory. Sanbonmatsu and coworkers have used a supercomputer to simulate a working ribosome, identifying eight new potential antibiotic target sites. "The study is the largest simulation performed to date in biology," Sanbonmatsu says.
Last year, Yonath, chemistry professor Lou Massa of Hunter College of the City University of New York, crystallographer Jerome Karle of the Naval Research Laboratory, Washington, D.C., and coworkers turned to density functional theory to model ribosome catalysis. They reported a quantum mechanical transition state for peptide bond formation in the ribosome. The study also "defined the activation energy of the reaction and identified ribosomal interactions that seem to stabilize the transition state, which is formed while the A-site tRNA is rotating into the P site," Yonath says.
Such revelations notwithstanding, the ribosome continues to hold onto a few secrets. "There have always been researchers who think that we understand how the ribosome works," Noller says. However, "at this point, in spite of high-resolution crystal structures and decades of biochemical, genetic, and biophysical studies, I don't think we understand the fundamental mechanisms at all," he says,
"How do tRNAs and mRNA move during translocation, a process that involves molecular movements of many tens of angstroms every 50 milliseconds or so?" Noller asks. "What is the role of EF-G in that process? How does EF-Tu speed up binding of aminoacyl-tRNA to the A site by several thousandfold? The ribosome is enormous and tremendously conserved phylogenetically, yet the things we claim to understand at this point involve only a handful of nucleotides."
"The questions are getting finer, and they're also getting harder to ask," Strobel notes. "Where one person says we have the answers, the next person says we have the questions."