Summary Phosphorylation is one of the most commonly used signaling mechanisms in biology. However, the molecular transition pathways between inactive and active states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of the signaling protein Nitrogen regulatory protein C (NtrC) by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding. We show that only a rarely populated, pre-existing active conformation is capable of being phosphorylated. Atomistic details of a pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009) is experimentally tested here by NMR dynamics experiments. We found that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results demonstrate the power of combining computation with experimental corroboration to unravel atomistic details of native-state protein energy landscapes by expanding the energy landscape from the ground states to transition landscapes.

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ⌬F508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL⅐CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

Capsids of many viruses assemble around nucleic acids or other polymers. Understanding how the properties of the packaged polymer affect the assembly process could promote biomedical efforts to prevent viral assembly or nanomaterials applications that exploit assembly. To this end, we simulate on a lattice the dynamical assembly of closed, hollow shells composed of several hundred to 1000 subunits, around a flexible polymer. We find that assembly is most efficient at an optimum polymer length that scales with the surface area of the capsid; polymers that are significantly longer than optimal often lead to partial-capsids with unpackaged polymer "tails" or a competition between multiple partial-capsids attached to a single polymer. These predictions can be tested with bulk experiments in which capsid proteins assemble around homopolymeric RNA or synthetic polyelectrolytes. We also find that the polymer can increase the net rate of subunit accretion to a growing capsid both by stabilizing the addition of new subunits and by enhancing the incoming flux of subunits; the effects of these processes may be distinguishable with experiments that monitor the assembly of individual capsids.

to distinguish from a state of true thermodynamic equilibrium. The pseudo LMA describes partitioning of capsid proteins between assembled capsids and a metastable, supersaturated solution of free proteins. This metastable state decays logarithmically slowly. We show that the line energy of assembly intermediates is the key control parameter of the pseudo LMA.