Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

No abstract

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an "all-or-none" transition (independent of how compact is the denatured state of a protein and independent of the protein-solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free-energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.

Folding of protein-like heteropolymers into unique 3D structures is investigated using Monte Carlo simulations on a cubic lattice. We found that folding time of chains of length N scales as N λ at temperature of fastest folding. For chains with random sequences of monomers λ ≈ 6, and for chains with sequences designed to provide a pronounced minimum of energy to their ground state conformation λ ≈ 4. Folding at low temperatures exhibits an Arrheniuslike behavior with the energy barrier E b ≈ φ|E n |, where E n is the energy of the native conformation. φ ≈ 0.18 both for random and designed sequences.

The implications of thermodynamics of heteropolymers for their folding kinetics are formulated and discussed. The predictions are tested by Monte Carlo simulation of folding of a lattice model 36-monomer proteins at different temperatures. Using a simulated annealing procedure in sequence space, a number of sequences are designed which have sufficiently low energy in a given target conformation. This conformation plays a role of the native structure for the model proteins. The folding transition is found to be cooperative, and the nature of the free energy barrier is studied. At high temperature the barrier is entropic, and at low temperature it is mainly energetic. This can be explained by transformation of the early partly folded intermediate from the disordered state (belonging to the quasicontinuous part of the energy spectrum) at high temperature to the non-native low-energy frozen conformation at low temperature. The latter plays a role of an off-pathway trap. A parallel folding process has been detected at low temperature where direct folding competes with relaxation of the intermediate. The frozen intermediate must unfold to make it possible to form a folding nucleus, which is the prerequisite of subsequent fast descent to the native state.