Folding mechanisms of individual beta-hairpins in a Go model of Pin1 WW domain by all-atom molecular dynamics simulations.
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ABSTRACT:
SUBMITTER: Luo Z
PROVIDER: S-EPMC2596927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We study the folding thermodynamics and kinetics of the Pin1 WW domain, a three-stranded beta-sheet protein, by using all-atom (except nonpolar hydrogens) discontinuous molecular dynamics simulations at various temperatures with a Gō model. The protein exhibits a two-state folding kinetics near the folding transition temperature. A good agreement between our simulations and the experimental measurements by the Gruebele group has been found, and the simulation sheds new insights into the structure of transition state, which is hard to be straightforwardly captured in experiments. The simulation also reveals that the folding pathways at approximately the transition temperature and at low temperatures are much different, and an intermediate state at a low temperature is predicted. The transition state of this small beta-protein at its folding transition temperature has a well-established hairpin 1 made of beta1 and beta2 strands while its low-temperature kinetic intermediate has a formed hairpin 2 composed of beta2 and beta3 strands. Theoretical results are compared with other simulation results as well as available experimental data. This study confirms that specific side-chain packing in an all-atom Gō model can yield a reasonable prediction of specific folding kinetics for a given protein. Different folding behaviors at different temperatures are interpreted in terms of the interplay of entropy and enthalpy in folding process.
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Project description:Engineered repeat proteins have proven to be a fertile ground for studying the competition between folding, misfolding and transient aggregation of tethered protein domains. We examine the interplay between folding and inter-domain interactions of engineered FiP35 WW domain repeat proteins with n = 1 through 5 repeats. We characterize protein expression, thermal and guanidium melts, as well as laser T-jump kinetics. All experimental data is fitted by a global fitting model with two states per domain (U, N), plus a third state M to account for non-native states due to domain interactions present in all but the monomer. A detailed structural model is provided by coarse-grained simulated annealing using the AWSEM Hamiltonian. Tethered FiP35 WW domains with n = 2 and 3 domains are just slightly less stable than the monomer. The n = 4 oligomer is yet less stable, its expression yield is much lower than the monomer's, and depends on the purification tag used. The n = 5 plasmid did not express at all, indicating the sudden onset of aggregation past n = 4. Thus, tethered FiP35 has a critical nucleus size for inter-domain aggregation of n ? 4. According to our simulations, misfolded structures become increasingly prevalent as one proceeds from monomer to pentamer, with extended inter-domain beta sheets appearing first, then multi-sheet 'intramolecular amyloid' structures, and finally novel motifs containing alpha helices. We discuss the implications of our results for oligomeric aggregate formation and structure, transient aggregation of proteins whilst folding, as well as for protein evolution that starts with repeat proteins.
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Project description:In this study, we apply a hybrid-resolution model, namely, PACE, to characterize the free energy surfaces (FESs) of Trp-cage and a WW-domain variant along with the respective folding mechanisms. Unbiased, independent simulations with PACE are found to achieve together multiple folding and unfolding events for both proteins, allowing us to perform network analysis of the FESs to identify folding pathways. PACE reproduces for both proteins expected complexity hidden in the folding FESs, in particular metastable non-native intermediates. Pathway analysis shows that some of these intermediates are, actually, on-pathway folding intermediates and that intermediates kinetically closest to the native states can be either critical on-pathway or off-pathway intermediates, depending on the protein. Apart from general insights into folding, specific folding mechanisms of the proteins are resolved. We find that Trp-cage folds via a dominant pathway in which hydrophobic collapse occurs before the N-terminal helix forms; full incorporation of Trp6 into the hydrophobic core takes place as the last step of folding, which, however, may not be the rate-limiting step. For the WW-domain variant studied, we observe two main folding pathways with opposite orders of formation of the two hairpins involved in the structure; for either pathway, formation of hairpin 1 is more likely to be the rate-limiting step. Altogether, our results suggest that PACE combined with network analysis is a computationally efficient and valuable tool for the study of protein folding.
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Project description:Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K(d) of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K(d) values for Fmoc-VPRpTPVGGGK-NH2 and Ac-VPRpTPV-NH2 were determined to be 36+/-4 and 110+/-30 microM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.
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Project description:We investigate the folding mechanism of the WW domain Fip35 using a realistic atomistic force field by applying the Dominant Reaction Pathways approach. We find evidence for the existence of two folding pathways, which differ by the order of formation of the two hairpins. This result is consistent with the analysis of the experimental data on the folding kinetics of WW domains and with the results obtained from large-scale molecular dynamics simulations of this system. Free-energy calculations performed in two coarse-grained models support the robustness of our results and suggest that the qualitative structure of the dominant paths are mostly shaped by the native interactions. Computing a folding trajectory in atomistic detail only required about one hour on 48 Central Processing Units. The gain in computational efficiency opens the door to a systematic investigation of the folding pathways of a large number of globular proteins.
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Project description:Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and/or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I' beta-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.
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Project description:Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.
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Project description:To benchmark RNA force fields, we compared the folding stabilities of three 12-nucleotide hairpin stem loops estimated by simulation to stabilities determined by experiment. We used umbrella sampling and a reaction coordinate of end-to-end (5' to 3' hydroxyl oxygen) distance to estimate the free energy change of the transition from the native conformation to a fully extended conformation with no hydrogen bonds between non-neighboring bases. Each simulation was performed four times using the AMBER FF99+bsc0+χOL3 force field, and each window, spaced at 1 Å intervals, was sampled for 1 μs, for a total of 552 μs of simulation. We compared differences in the simulated free energy changes to analogous differences in free energies from optical melting experiments using thermodynamic cycles where the free energy change between stretched and random coil sequences is assumed to be sequence-independent. The differences between experimental and simulated ΔΔ G° are, on average, 0.98 ± 0.66 kcal/mol, which is chemically accurate and suggests that analogous simulations could be used predictively. We also report a novel method to identify where replica free energies diverge along a reaction coordinate, thus indicating where additional sampling would most improve convergence. We conclude by discussing methods to more economically perform these simulations.
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Project description:We study the folding mechanism of a triple beta-strand WW domain from the Formin binding protein 28 (FBP28) at atomic resolution with explicit water model using replica exchange molecular dynamics computer simulations. Extended sampling over a wide range of temperatures to obtain the free energy, enthalpy, and entropy surfaces as a function of structural coordinates has been performed. Simulations were started from different configurations covering the folded and unfolded states. In the free energy landscape a transition state is identified and its structures and -values are compared with experimental data from a homologous protein, the prolyl-isomerase Pin1 WW domain. A stable intermediate state is found to accumulate during the simulation characterized by the carboxyl-terminal beta-strand 3 having misregistered hydrogen bonds and where the structural heterogeneity is due to nonnative turn II formation. Furthermore, the aggregation behavior of the FBP28 WW domain may be related to one such misfolded structure, which has a much lower free energy of dimer formation than that of the native dimer. Based on the misfolded dimer, aggregation to form protofibril structure is discussed.
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Project description:Reaching the native states of small proteins, a necessary step towards a comprehensive understanding of the folding mechanisms, has remained a tremendous challenge to ab initio protein folding simulations despite the extensive effort. In this work, the folding process of the B domain of protein A (BdpA) has been simulated by both conventional and replica exchange molecular dynamics using AMBER FF03 all-atom force field. Started from an extended chain, a total of 40 conventional (each to 1.0 micros) and two sets of replica exchange (each to 200.0 ns per replica) molecular dynamics simulations were performed with different generalized-Born solvation models and temperature control schemes. The improvements in both the force field and solvent model allowed successful simulations of the folding process to the native state as demonstrated by the 0.80 A C(alpha) root mean square deviation (RMSD) of the best folded structure. The most populated conformation was the native folded structure with a high population. This was a significant improvement over the 2.8 A C(alpha) RMSD of the best nativelike structures from previous ab initio folding studies on BdpA. To the best of our knowledge, our results demonstrate, for the first time, that ab initio simulations can reach the native state of BdpA. Consistent with experimental observations, including Phi-value analyses, formation of helix II/III hairpin was a crucial step that provides a template upon which helix I could form and the folding process could complete. Early formation of helix III was observed which is consistent with the experimental results of higher residual helical content of isolated helix III among the three helices. The calculated temperature-dependent profile and the melting temperature were in close agreement with the experimental results. The simulations further revealed that phenylalanine 31 may play critical to achieve the correct packing of the three helices which is consistent with the experimental observation. In addition to the mechanistic studies, an ab initio structure prediction was also conducted based on both the physical energy and a statistical potential. Based on the lowest physical energy, the predicted structure was 2.0 A C(alpha) RMSD away from the experimentally determined structure.
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