Exploring the free energy landscape of proteins and modeling the corresponding functional aspects presents a major challenge for computer simulation approaches. This challenge is due to the complexity of the landscape and the enormous computer time needed for converging simulations. The use of various simplified coarse grained (CG) models offers an effective way of sampling the landscape, but most current models are not expected to give a reliable description of protein stability and functional aspects. The main problem is associated with insufficient focus on the electrostatic features of the model. In this respect our recent CG model offers significant advantage as it has been refined while focusing on its electrostatic free energy. Here we review the current state of our model, describing recent refinement, extensions and validation studies while focusing on demonstrating key applications. These include studies of protein stability, extending the model to include membranes and electrolytes and electrodes as well as studies of voltage activated proteins, protein insertion trough the translocon, the action of molecular motors and even the coupling of the stalled ribosome and the translocon. Our example illustrates the general potential of our approach in overcoming major challenges in studies of structure function correlation in proteins and large macromolecular complexes.

The understanding of the mechanism of insertion of transmembrane (TM) helixes through the translocon presents a major open challenge. Although the experimental information about the partition of the inserted helices between the membrane and the solution contains crucial information about this process, it is not clear how to extract this information. In particular, it is not clear how to rationalize the small apparent insertion energy, ΔG app , of an ionized residue in the center of a TM helix. Here we explore the nature of the insertion energies, asking what should be the value of these parameters if their measurements represent equilibrium conditions. This is done using a coarse-grained model with advanced electrostatic treatment. Estimating the energetics of ionized arginine of a TM helix in the presence of neighboring helixes or the translocon provides a rationale for the observed ΔG app of ionized residues. It is concluded that the apparent insertion free energy of TM with charged residues reflects probably more than just the free energy of moving the isolate single helix from water into the membrane. The present approach should be effective not only in exploring the mechanism of the operation of the translocon but also for studies of other membrane proteins.electrostatics computer simulations | simplified models T he insertion of transmembrane (TM) proteins into membranes is a subject of great current interest (1-6). It is known that the recognition of proteins is performed by the translocon complex that ensures both the translocation of globular proteins across membranes and the integration of membrane proteins into membranes (7). Biochemical studies have provided major information about the insertion process, and structural studies have provided key hints about the actual insertion mechanism (1,(8)(9)(10). Furthermore, clever experiments by von Heijne, White, and their coworkers (5) have determined a scale that reflects the apparent energetic of the inserting a TM helix into a membrane in biological conditions. These workers took the sequence of the double-spanning protein (bacterial leader peptidase) and added to two TM helixes of this protein (TM1 and TM2) an additional helix (the H helix), which was flanked by two acceptor sites for N-linked glycosylation (5). The degree of membrane integration of the H helix was then analyzed by the number of glycosylated sites, and the apparent equilibrium constants, K app ¼ f 1g ∕f 2g (where f 1g and f 2g were determined by the fractions of singly and doubly glycosylated proteins, respectively), were calculated. These values were then converted to the relevant apparent free energies, ΔG app ¼ −RT ln K app . Decomposing ΔG app to the contribution from different amino acids provided the insertion scale for the H helices, with each of the 20 naturally occurring amino acids placed in the middle of the 19-residue hydrophobic stretch (see ref. 5 and Results for more details).The experimental determination of the "biological" hydrophobic scale (5) challenges one to rationa...

The elucidation of the molecular nature of the translocon-assisted protein insertion is a challenging problem due to the complexity of this process. Furthermore, the limited availability of crucial structural information makes it hard to interpret the hints about the insertion mechanism provided by biochemical studies. At present, it is not practical to explore the insertion process by brute force simulation approaches due to the extremely lengthy process and very complex landscape. Thus, this work uses our previously developed coarse-grained model and explores the energetics of the membrane insertion and translocation paths. The trend in the calculated free-energy profiles is verified by evaluating the correlation between the calculated and observed effect of mutations as well as the effect of inverting the signal peptide that reflects the "positive-inside" rule. Furthermore, the effect of the tentative opening induced by the ribosome is found to reduce the kinetic barrier. Significantly, the trend of the forward and backward energy barriers provides a powerful way to analyze key energetics information. Thus, it is concluded that the insertion process is most likely a nonequilibrium process. Moreover, we provided a general formulation for the analysis of the elusive apparent membrane insertion energy, ΔG app , and conclude that this important parameter is unlikely to correspond to the freeenergy difference between the translocon and membrane. Our formulation seems to resolve the controversy about ΔG app for Arg.coarse-grain modeling | hydrophobicity scale | topology T he establishment of the correct functional topology of membrane proteins is a subject of great current interest (e.g., refs. 1-3). It is known that the protein-conducting channel named translocon (TR) plays a vital role in membrane proteins biogenesis (4). Although biochemical and structural (e.g., refs. 5 and 6) studies have provided crucial information about the insertion process, the understanding of this process is still limited. The difficulties in gaining detailed understanding are also apparent from the emerging problem in fully defining the molecular meaning of the intriguing results about the apparent free energy, ΔG app . That is, the concept of ΔG app , introduced by Hessa et al. (7) for the assessment of inserted versus secreted helical domains (Background), appeared in recent years to be more complex than previously thought. Apparently, the most logical implication of the original descriptions of ΔG app has been that it represents an equilibrium result of the partition between membrane and water. In fact, this was implied by the attempts to correlate ΔG app with the water-membrane partitions. However, different works (8-10) implied that the corresponding equilibrium constant corresponds to the equilibrium between the TR and the membrane (see also Background). Unfortunately, none of these works has offered a clear physical rationale for why one has to consider such equilibrium without considering the transfer to water. We note that eve...

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein.We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/ Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to welldefined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.functional genomics | PTEN | tumor suppressor

The nature of the coupling between the stalling of the elongated nascent peptide chain in the ribosome and its insertion through the translocon is analyzed, focusing on the recently discovered biphasic force that overcomes the stalling barrier. The origin of this long-range coupling is explored by coarse-grained simulations that combine the translocon (TR) insertion profile and the effective chemical barrier for the extension of the nascent chain in the ribosome. Our simulation determined that the inserted H segment is unlikely to climb the TR barrier in parallel with the peptide synthesis chemical step and that the nascent chain should first overcome the chemical barriers and move into the ribosome-TR gap region before the insertion into the TR tunnel. Furthermore, the simulations indicate that the coupled TR-chemistry free energy profile accounts for the biphasic force. Apparently, although the overall elongation/insertion process can be depicted as a tug-ofwar between the forces of the TR and the ribosome, it is actually a reflection of the combined free-energy landscape. Most importantly, the present study helps to relate the experimental observation of the biphasic force to crucial information about the elusive path and barriers of the TR insertion process.he elongation of nascent peptide chains during their synthesis by the ribosome and the translocon (TR)-assisted insertion of the generated chain into the membrane are coupled in an intriguing way. A glimpse into this coupling has been provided recently by the von Heine group (1), who observed an interplay between the stalling of the elongation process and the TR insertion process. More specifically, it was found that in cases when the elongation stalls due to the presence of an RXXP-type sequence, the process is reactivated by the force generated in the TR for different lengths of the inserted chain. While this finding is very exciting, it is not clear what the exact origin of the applied force is and how it can be coupled to the elongation process, where the insertion into the TR involves an uphill penetration process.The peptide elongation process and the subsequent insertion are described schematically in Fig. 1 for the system studied by von Heijne and coworkers (1). As seen from the figure, after the peptide bond is synthesized, the extended peptide moves through the TR and then inserts into the membrane. However, the chain extension process can be stalled during the elongation process of the RXXP and other sequences (2-6). This stall reflects, most probably, the increase in the activation barrier for the peptide bond formation, due in part to changes in the preorganization of the active site (SI Text). As demonstrated in ref. 1, for some lengths of the linker (denoted by L) the stalling is released as the H segment (a 19-residue leucine-alanine-based peptide, which was introduced into the leader peptidase protein) passes through the TR (Fig. 1 B and C). On the other hand, for other lengths the stalling cannot be overcome and the ribosome remains atta...