HIV-1 protease (PR) is an effective drug target for antiviral inhibitors. The conformational dynamics in the flaps of HIV-1 PR plays a crucial role in the mechanism of substrate binding. Here, the structural properties of the functionally important intermediate states of the flap opening transition of HIV-1 PR have been characterized by enhanced sampling molecular dynamics simulation as well as long-time conventional non-enhanced simulations at atomic level. Not only crystallographically measured “closed” and “semi-open” structures but also a novel “curled” structure of HIV-1 PR is captured by both kinds of simulations qualitatively and quantitatively. The observation of the “curled” intermediate state helps to connect all other functionally important states to provide an integrated view of the transition pathway of the flap opening of HIV-1 PR (closed → curled → semi-open → fully open). The key residue–residue interactions which are broken or formed in the transition are analyzed to reveal the inherent driving force for the protein conformational transition.

B-RAF protein kinase is a promising target to treat malignant melanoma. The kinase activity of B-RAF is regulated by a “DFG-flip” conformational transition between functional DFG-in and DFG-out states. The difficulty in resolving the activation loop in crystal structures and the even greater difficulty in experimentally capturing high-energy-level transient structures render elusive the molecular mechanism of the B-RAF functional conformational transition. Here, a homology modeling technique and an enhanced sampling molecular dynamics simulation were used to identify and energetically characterize the conformational transition pathway of B-RAF on a multi-dimensional free-energy landscape. The results reveal that the conformational transition is a two-state transition, with the evaluated free-energy barrier comparable to those of other kinds of kinases as reported in the previous literature. Hydrophobic interactions between activation loop and neighboring segments are suggested to dominate the conformational transition and determine the free-energy barrier. The detailed analysis of hydrophobic interactions involved in the conformational transition may show a suitable pathway for the development of the B-RAF inhibitor.

Integrated-tempering-sampling molecular dynamics simulation is utilized to investigate the folding of a 67-residue three-α-helix bundle, α3W. Reversible folding and unfolding can be observed in individual trajectories and a total of 28 folding events are achieved within 7 μs simulation, giving sufficient sampling on the configuration space of protein folding. The native-like state with a left-handed topology constitutes the largest fraction of the conformational ensemble sampled by the simulation. In addition, a misfolded state with mirror-image (right-handed) topology is observed with smaller population. The free energy landscape analysis demonstrates that the folding of α3W is initiated by the formation of α-helical secondary structures and is followed by the assembling of folded α-helices to construct tertiary structure. The “correct” α-helix assembling which leads to the native structure is mainly dominated by inter-helical hydrophobic interactions whereas the “incorrect” assembling which leads to a misfolded mirror-image structure is highly affected by not only hydrophobic but also charge interactions. It is speculated on the basis of the present study on α3W and other studies on the B domain of protein A and α3D that the misfolding probability of α-helix bundle proteins is dependent on the strength of intra-protein hydrophobic and charge interactions: proteins containing stronger hydrophobic interactions but weaker charge interactions should have smaller misfolding probability. The importance of intra-protein hydrophobic interactions in preventing protein misfolding has been also seen in our previous studies on β-hairpins. Therefore, the present study along with our previous studies provide comprehensive, atomic-level picture of the folding/misfolding of α-helix bundle and β-hairpin proteins.

Proteins fold through complex inter-residue interactions which are mutually supportive and cooperatively lead to thermodynamically favorable native structures. Competing (misfolded) structures, however, could exist, which might affect the thermodynamic and kinetic properties of folded structure. Running long-time REMD simulations on two β-structured polypeptides, the present study identifies the folded and (less populated) competing misfolded states of β-hairpins. Of particular interest is a one-residue shifted misfolded state which has been often seen in previous reports. The folding and misfolding pathways are then energetically characterized by free energy landscape analysis, indicating that the folding and misfolding of β-hairpin are parallel pathways and a protein’s selection of following which pathway is a consequence of the competition between the formation of alterable turn configurations and cross-strand hydrophobic interactions. Proteins possessing high percentage of hydrophobic residues introduce strong cross-strand hydrophobic interactions which stabilize the native structural elements in the folding pathway, leading to low possibility of misfolding. The present study provides novel insights into the origin of sequence-dependent β-hairpin misfolding “hidden” behind experimentally detectable β-hairpin folding, suggesting the direction for the structure design of β-structured protein.

Mixtures of osmolytes are present in the cell. Therefore, the understanding of the interplay of mixed osmolyte molecules and their combined effects on protein structure is of fundamental importance. In this article, the structure stability of a model protein (BdpA) in the mixture of guanidinium thiocyanate (GdmSCN) and methanol (MeOH) was investigated by molecular dynamics simulation. It was observed that guanidinium (Gdm+) is driven to protein surface by favorable electrostatic interactions and MeOH is driven by both favorable electrostatic and VDW interactions, respectively. The mixture of Gdm+ and MeOH doesnot affect the electrostatic energy distribution of Gdm+ but does reduce the difference in VDW energy of MeOH between the regions of protein surface and bulk solution. As a result, the accumulation level of Gdm+ is not influenced, but the accumulation level of MeOH is lowered in mixed solution. The tertiary structure stability of protein is determined by the accumulated strength of VDW interactions from MeOH to protein side chain, and the secondary structure stability is correlated to the strength of combined electrostatic energies from solvent (water) and cosolvent (Gdm+ and MeOH) to protein backbone, particularly in hydrogen bonding part. The mixture of GdmSCN with low-concentrated MeOH stabilizes native structure of BdpA whereas the further increase of MeOH concentration denatures native structure of protein to expanded unfolded structure. The present study together with our previous study on the mixture of GdmSCN and 2,2,2-trifluoroethanol (TFE) provides novel insights into the effects of mixed osmolytes on protein structure.

Large-scale conformational changes of proteins are usually associated with the binding of ligands. Because the conformational changes are often related to the biological functions of proteins, understanding the molecular mechanisms of these motions and the effects of ligand binding becomes very necessary. In the present study, we use the combination of normal-mode analysis and umbrella sampling molecular dynamics simulation to delineate the atomically detailed conformational transition pathways and the associated free-energy landscapes for three well-known protein systems, viz., adenylate kinase (AdK), calmodulin (CaM), and p38α kinase in the absence and presence of respective ligands. For each protein under study, the transient conformations along the conformational transition pathway and thermodynamic observables are in agreement with experimentally and computationally determined ones. The calculated free-energy profiles reveal that AdK and CaM are intrinsically flexible in structures without obvious energy barrier, and their ligand binding shifts the equilibrium from the ligand-free to ligand-bound conformation (population shift mechanism). In contrast, the ligand binding to p38α leads to a large change in free-energy barrier (ΔΔG ≈ 7 kcal/mol), promoting the transition from DFG-in to DFG-out conformation (induced fit mechanism). Moreover, the effect of the protonation of D168 on the conformational change of p38α is also studied, which reduces the free-energy difference between the two functional states of p38α and thus further facilitates the conformational interconversion. Therefore, the present study suggests that the detailed mechanism of ligand binding and the associated conformational transition is not uniform for all kinds of proteins but correlated to their respective biological functions.

A comparative study on the folding of multiple three-α-helix bundle proteins including α3D, α3W, and the B domain of protein A (BdpA) is presented. The use of integrated-tempering-sampling molecular dynamics simulations achieves reversible folding and unfolding events in individual short trajectories, which thus provides an efficient approach to sufficiently sample the configuration space of protein and delineate the folding pathway of α-helix bundle. The detailed free energy landscape analyses indicate that the folding mechanism of α-helix bundle is not uniform but sequence dependent. A simple model is then proposed to predict folding mechanism of α-helix bundle on the basis of amino acid composition: α-helical proteins containing higher percentage of hydrophobic residues than charged ones fold via nucleation–condensation mechanism (e.g., α3D and BdpA) whereas proteins having opposite tendency in amino acid composition more likely fold via the framework mechanism (e.g., α3W). The model is tested on various α-helix bundle proteins, and the predicted mechanism is similar to the most approved one for each protein. In addition, the common features in the folding pathway of α-helix bundle protein are also deduced. In summary, the present study provides comprehensive, atomic-level picture of the folding of α-helix bundle proteins.

The effects of intrinsic structural flexibility of calmodulin protein on the mechanism of its allosteric conformational transition are investigated in this article. Using a novel in silico approach, the conformational transition pathways of intact calmodulin as well as the isolated N- and C- terminal domains are identified and energetically characterized. It is observed that the central α-helix linker amplifies the structural flexibility of intact Ca2+-free calmodulin, which might facilitate the transition of the two domains. As a result, the global conformational transition of Ca2+-free calmodulin is initiated by the barrierless transition of two domains and proceeds through the barrier associated unwinding and bending of the central α-helix linker. The binding of Ca2+ cations to calmodulin further increases the structural flexibility of the C-terminal domain and results in a downhill transition pathway of which all regions transit in a concerted manner. On the other hand, the separation of the N- and C-terminal domains from calmodulin protein loses the mediating function of central α-helix linker, leading to more difficult conformational transitions of both domains. The present study provides novel insights into the correlation of the integrity of protein, the structural flexibility, and the mechanism of conformational transition of proteinlike calmodulin.

B-RAF protein kinase is a promising target to treat malignant melanoma. The kinase activity of B-RAF is regulated by a “DFG-flip” conformational transition between functional DFG-in and DFG-out states. The difficulty in resolving the activation loop in crystal structures and the even greater difficulty in experimentally capturing high-energy-level transient structures render elusive the molecular mechanism of the B-RAF functional conformational transition. Here, a homology modeling technique and an enhanced sampling molecular dynamics simulation were used to identify and energetically characterize the conformational transition pathway of B-RAF on a multi-dimensional free-energy landscape. The results reveal that the conformational transition is a two-state transition, with the evaluated free-energy barrier comparable to those of other kinds of kinases as reported in the previous literature. Hydrophobic interactions between activation loop and neighboring segments are suggested to dominate the conformational transition and determine the free-energy barrier. The detailed analysis of hydrophobic interactions involved in the conformational transition may show a suitable pathway for the development of the B-RAF inhibitor.