Hydrogen bonding heterogeneity correlates with protein folding transition state passage time as revealed by data sonification.

Protein-protein and protein-water hydrogen bonding interactions play essential roles in the way a protein passes through the transition state during folding or unfolding, but the large number of these interactions in molecular dynamics (MD) simulations makes them difficult to analyze. Here, we introduce a state space representation and associated "rarity" measure to identify and quantify transition state passage (transit) events. Applying this representation to a long MD simulation trajectory that captured multiple folding and unfolding events of the GTT WW domain, a small protein often used as a model for the folding process, we identified three transition categories: Highway (faster), Meander (slower), and Ambiguous (intermediate). We developed data sonification and visualization tools to analyze hydrogen bond dynamics before, during, and after these transition events. By means of these tools, we were able to identify characteristic hydrogen bonding patterns associated with "Highway" versus "Meander" versus "Ambiguous" transitions and to design algorithms that can identify these same folding pathways and critical protein-water interactions directly from the data. Highly cooperative hydrogen bonding can either slow down or speed up transit. Furthermore, an analysis of protein-water hydrogen bond dynamics at the surface of WW domain shows an increase in hydrogen bond lifetime from folded to unfolded conformations with Ambiguous transitions as an outlier. In summary, hydrogen bond dynamics provide a direct window into the heterogeneity of transits, which can vary widely in duration (by a factor of 10) due to a complex energy landscape.

In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non-native interactions.

We examine the influence of cellular interactions in all-atom models of a section of the Homo sapiens cytoplasm on the early folding events of the three-helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30-microsecond simulations. We did however capture the formation of all three helices in a compact native-like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta-sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild-type sequence. This study demonstrates that even fast-folding proteins can get stuck in non-native states in the cell, making them useful models for protein-chaperone interactions and early stages of aggregate formation relevant to cellular disease.

TMAO: Protecting proteins from feeling the heat.

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 µs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules "herding" water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

Proteins: "Boil 'Em, Mash 'Em, Stick 'Em in a Stew".

Cells of the vast majority of organisms are subject to temperature, pressure, pH, ionic strength, and other stresses. We discuss these effects in the light of protein folding and protein interactions in vitro, in complex environments, in cells, and in vivo. Protein phase diagrams provide a way of organizing different structural ensembles that occur under stress and how one can move among ensembles. Experiments that perturb biomolecules in vitro or in cells by stressing them have revealed much about the underlying forces that are competing to control protein stability, folding, and function. Two phenomena that emerge and serve to broadly classify effects of the cellular environment are crowding (mainly due to repulsive forces) and sticking (mainly due to attractive forces). The interior of cells is closely balanced between these emergent effects, and stress can tip the balance one way or the other. The free energy scale involved is small but significant on the scale of the "on/off switches" that control signaling in cells or of protein-protein association with a favorable function such as increased enzyme processivity. Quantitative tools from biophysical chemistry will play an important role in elucidating the world of crowding and sticking under stress.

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression.

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.

Critical phenomena in the temperature-pressure-crowding phase diagram of a protein.

In the cell, proteins fold and perform complex functions through global structural rearrangements. Function requires a protein to be at the brink of stability to be susceptible to small environmental fluctuations, yet stable enough to maintain structural integrity. These apparently conflicting behaviors are exhibited by systems near a critical point, where distinct phases merge-a concept beyond previous studies indicating proteins have a well-defined folded/unfolded phase boundary in the pressure-temperature plane. Here, by modeling the protein phosphoglycerate kinase (PGK) on the temperature (T), pressure (P), and crowding volume-fraction (ϕ) phase diagram, we demonstrate a critical transition where phases merge, and PGK exhibits large structural fluctuations. Above the critical point, the difference between the intermediate and unfolded phases disappears. When ϕ increases, the critical point moves to lower T c. We verify the calculations with experiments mapping the T-P-ϕ space, which likewise reveal a critical point at 305 K and 170 MPa that moves to lower T c as ϕ increases. Crowding places PGK near a critical line in its natural parameter space, where large conformational changes can occur without costly free energy barriers. Specific structures are proposed for each phase based on simulation.