Background

An important element in homology modeling is the use of rotamers to parameterize the sidechain conformation. Despite the many libraries of sidechain rotamers that have been developed, a number of rotamers have been overlooked, due to the fact that they involve hydrogen atoms.

Results

We identify new, well-populated rotamers that involve the hydroxyl-hydrogen atoms of Ser, Thr and Tyr, and the sulfhydryl-hydrogen atom of Cys, using high-resolution crystal structures (<1.2 A). Although there were refinement artifacts in these structures, comparison with the electron-density maps allowed the placement of hydrogen atoms involved in hydrogen bonds. The chi2 rotamers in Ser, Thr and Cys are consistent with tetrahedral bonding, while the chi3 rotamers in Tyr are consistent with trigonal-planar bonding. Similar rotamers are found in hydrogen atoms that were computationally placed with the Reduce program from the Richardson lab.

Conclusion

Knowledge of these new rotamers will improve the evaluation of hydrogen-bonding networks in protein structures.

Peptides often have conformational preferences. We simulated 133 peptide 8-mer fragments from six different proteins, sampled by replica-exchange molecular dynamics using Amber7 with a GB/SA (generalized-Born/solvent-accessible electrostatic approximation to water) implicit solvent. We found that 85 of the peptides have no preferred structure, while 48 of them converge to a preferred structure. In 85% of the converged cases (41 peptides), the structures found by the simulations bear some resemblance to their native structures, based on a coarse-grained backbone description. In particular, all seven of the beta hairpins in the native structures contain a fragment in the turn that is highly structured. In the eight cases where the bioinformatics-based I-sites library picks out native-like structures, the present simulations are largely in agreement. Such physics-based modeling may be useful for identifying early nuclei in folding kinetics and for assisting in protein-structure prediction methods that utilize the assembly of peptide fragments.

One of the applications of Molecular Dynamics (MD) simulations is to explore the energetic barriers to mechanical unfolding of proteins such as occurs in response to the mechanical pulling of single molecules in Atomic Force Microscopy (AFM) experiments. Although Steered Molecular Dynamics simulations have provided microscopic details of the unfolding process during the pulling, the simulated forces required for unfolding are typically far in excess of the measured values. To rectify this, we have developed the Pulsed Unconstrained Fluctuating Forces (PUFF) method, which induces constant-momentum motions by applying forces directly to the instantaneous velocity of selected atoms in a protein system. The driving forces are applied in pulses, which allows the system to relax between pulses, resulting in more accurate unfolding force estimations than in previous methods. In the cases of titin, ubiquitin and e2lip3, the PUFF trajectories produce force fluctuations that agree quantitatively with AFM experiments. Another useful property of PUFF is that simulations get trapped if the target momentum is too low, simplifying the discovery and analysis of unfolding intermediates.

Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease alpha-lytic protease (alphaLP) has done just that, as its unfolding is both very slow (t(1/2) approximately 1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to alphaLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the alphaLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both alphaLP and trypsin. The simulated alphaLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that alphaLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of alphaLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases.