Biogenesis, quality control, and structural dynamics of proteins as explored in living cells via site-directed photocrosslinking.

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the site-directed photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K.

To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.

Equilibrium transitions between side-chain conformations in leucine and isoleucine.

Despite recent improvements in computational methods for protein design, we still lack a quantitative, predictive understanding of the intrinsic probabilities for amino acids to adopt particular side-chain conformations. Surprisingly, this question has remained unsettled for many years, in part because of inconsistent results from different experimental approaches. To explicitly determine the relative populations of different side-chain dihedral angles, we performed all-atom hard-sphere Langevin Dynamics simulations of leucine (Leu) and isoleucine (Ile) dipeptide mimetics with stereo-chemical constraints and repulsive-only steric interactions between non-bonded atoms. We determine the relative populations of the different χ(1) and χ(2) dihedral angle combinations as a function of the backbone dihedral angles ϕ and ψ. We also propose, and test, a mechanism for inter-conversion between the different side-chain conformations. Specifically, we discover that some of the transitions between side-chain dihedral angle combinations are very frequent, whereas others are orders of magnitude less frequent, because they require rare coordinated motions to avoid steric clashes. For example, to transition between different values of χ(2), the Leu side-chain bond angles κ(1) and κ(2) must increase, whereas to transition in χ(1), the Ile bond angles λ(1) and λ(2) must increase. These results emphasize the importance of computational approaches in stimulating further experimental studies of the conformations of side-chains in proteins. Moreover, our studies emphasize the power of simple steric models to inform our understanding of protein structure, dynamics, and design.

Recent applications of boxed molecular dynamics: a simple multiscale technique for atomistic simulations.

In this paper, we briefly review the boxed molecular dynamics (BXD) method which allows analysis of thermodynamics and kinetics in complicated molecular systems. BXD is a multiscale technique, in which thermodynamics and long-time dynamics are recovered from a set of short-time simulations. In this paper, we review previous applications of BXD to peptide cyclization, solution phase organic reaction dynamics and desorption of ions from self-assembled monolayers (SAMs). We also report preliminary results of simulations of diamond etching mechanisms and protein unfolding in atomic force microscopy experiments. The latter demonstrate a correlation between the protein's structural motifs and its potential of mean force. Simulations of these processes by standard molecular dynamics (MD) is typically not possible, because the experimental time scales are very long. However, BXD yields well-converged and physically meaningful results. Compared with other methods of accelerated MD, our BXD approach is very simple; it is easy to implement, and it provides an integrated approach for simultaneously obtaining both thermodynamics and kinetics. It also provides a strategy for obtaining statistically meaningful dynamical results in regions of configuration space that standard MD approaches would visit only very rarely.