Intrinsically disordered proteins (IDPs) adopt heterogeneous ensembles of conformations under physiological conditions. Understanding the relationship between amino acid sequence and conformational ensembles of IDPs can help clarify the role of disorder in physiological function. Recent studies revealed that polar IDPs favor collapsed ensembles in water despite the absence of hydrophobic groups-a result that holds for polypeptide backbones as well. By studying highly charged polypeptides, a different archetype of IDPs, we assess how charge content modulates the intrinsic preference of polypeptide backbones for collapsed structures. We characterized conformational ensembles for a set of protamines in aqueous milieus using molecular simulations and fluorescence measurements. Protamines are arginine-rich IDPs involved in the condensation of chromatin during spermatogenesis. Simulations based on the ABSINTH implicit solvation model predict the existence of a globule-to-coil transition, with net charge per residue serving as the discriminating order parameter. The transition is supported by quantitative agreement between simulation and experiment. Local conformational preferences partially explain the observed trends of polymeric properties. Our results lead to the proposal of a schematic protein phase diagram that should enable prediction of polymeric attributes for IDP conformational ensembles using easily calculated physicochemical properties of amino acid sequences. Although sequence composition allows the prediction of polymeric properties, interresidue contact preferences of protamines with similar polymeric attributes suggest that certain details of conformational ensembles depend on the sequence. This provides a plausible mechanism for specificity in the functions of IDPs.Monte Carlo | polyampholyte | polyelectrolyte I ntrinsically disordered proteins (IDPs) are a class of proteins that fail to fold autonomously in aqueous solutions to welldefined three-dimensional structures (1, 2). This "intrinsic disorder" has been implicated in a range of regulatory functions that require IDPs to interact with other macromolecular ligands (3-11). Many of these interactions promote disorder-to-order transitions within IDPs (3, 9), and different mechanistic models (3, 12, 13) have been proposed for coupled folding and binding. To develop a better understanding of how disorder is used in function (2), we have pursued quantitative, polymer-physics-based descriptions (14-16) for conformational ensembles of .Low hydrophobicity is a defining characteristic of IDP sequences (21,22). This suggests that IDPs cannot collapse to form compact, globular conformations in aqueous solutions (21). However, spectroscopic (23-27) and computational investigations (17, 28) have shown that polar tracts form heterogeneous ensembles of collapsed structures in aqueous solutions. These sequences are rich in uncharged, polar amino acids and are devoid of canonical hydrophobic residues. Collapse of polar tracts has been observed for polyglutamine (17...

Intrinsically disordered proteins (IDPs) are functional proteins that do not fold into well-defined three-dimensional structures under physiological conditions. IDP sequences have low hydrophobicity, and hence, recent experiments have focused on quantitative studies of conformational ensembles of archetypal IDP sequences such as polyglutamine and glycine-serine block copolypeptides. Results from these experiments show that, despite the absence of hydrophobic residues, polar IDPs prefer ensembles of collapsed structures in aqueous milieus. Do these preferences originate in interactions that are unique to polar sidechains? The current study addresses this issue by analyzing conformational equilibria for polyglycine and a glycine-serine block copolypeptide in two environments, namely, water and 8 M urea. Polyglycine, a poly secondary-amide, has no sidechains and is a useful model system for generic polypeptide backbones. Results based on large-scale molecular dynamics simulations show that polyglycine forms compact, albeit disordered, globules in water and swollen, disordered coils in 8 M urea. There is minimal overlap between conformational ensembles in the two environments. Analysis of order parameters derived from theories for flexible polymers show that water at ambient temperatures is a poor solvent for generic polypeptide backbones. Therefore, the experimentally observed preferences for polyglutamine and glycine-serine block copolypeptides must originate, at least partially, in polypeptide backbones. A preliminary analysis of the driving forces that lead to distinct conformational preferences for polyglycine in two different environments is presented. Implications for describing conformational ensembles of generic IDP sequences are also discussed.

Synopsis Intrinsically disordered proteins participate in important protein-protein and protein-nucleic acid interactions and control cellular phenotypes through their prominence as dynamic organizers of transcriptional, post-transcriptional, and signaling networks. These proteins challenge the tenets of the structure-function paradigm and their functional mechanisms remain a mystery given that they fail to fold autonomously into specific structures. Solving this mystery requires a first principles understanding of the quantitative relationships between information encoded in the sequences of disordered proteins and the ensemble of conformations they sample. Advances in quantifying sequence-ensemble relationships have been facilitated through a four-way synergy between bioinformatics, biophysical experiments, computer simulations, and polymer physics theories. Here, we review these advances and the resultant insights that allow us to develop a concise quantitative framework for describing sequence-ensemble relationships of intrinsically disordered proteins.

Accurate models of alkali and halide ions in aqueous solution are necessary for computer simulations of a broad variety of systems. Previous efforts to develop ion force fields have generally focused on reproducing experimental measurements of aqueous solution properties such as hydration free energies and ion-water distribution functions. This dependency limits transferability of the resulting parameters because of the variety and known limitations of water models. We present a solvent-independent approach to calibrating ion parameters based exclusively on crystal lattice properties. Our procedure relies on minimization of lattice sums to calculate lattice energies and interionic distances instead of equilibrium ensemble simulations of dense fluids. The gain in computational efficiency enables simultaneous optimization of all parameters for Li+, Na+, K+, Rb+, Cs+, F-, Cl-, Br-, and I- subject to constraints that enforce consistency with periodic table trends. We demonstrate the method by presenting lattice-derived parameters for the primitive model and the Lennard-Jones model with Lorentz-Berthelot mixing rules. The resulting parameters successfully reproduce the lattice properties used to derive them and are free from the influence of any water model. To assess the transferability of the Lennard-Jones parameters to aqueous systems, we used them to estimate hydration free energies and found that the results were in quantitative agreement with experimentally measured values. These lattice-derived parameters are applicable in simulations where coupling of ion parameters to a particular solvent model is undesirable. The simplicity and low computational demands of the calibration procedure make it suitable for parametrization of crystallizable ions in a variety of force fields.

Poly-L-proline (PLP) polymers are useful mimics of biologically relevant proline-rich sequences. Biophysical and computational studies of PLP polymers in aqueous solutions are challenging because of the diversity of length scales and the slow time scales for conformational conversions. We describe an atomistic simulation approach that combines an improved ABSINTH implicit solvation model, with conformational sampling based on standard and novel Metropolis Monte Carlo moves. Refinements to forcefield parameters were guided by published experimental data for proline-rich systems. We assessed the validity of our simulation results through quantitative comparisons to experimental data that were not used in refining the forcefield parameters. Our analysis shows that PLP polymers form heterogeneous ensembles of conformations characterized by semi-rigid, rod-like segments interrupted by kinks, which result from a combination of internal cis peptide bonds, flexible backbone ψ-angles, and the coupling between ring puckering and backbone degrees of freedom.

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Eukaryotic proteins often possess long stretches that fail to adopt well-defined, three-dimensional structures. These intrinsically disordered regions are associated with cell signaling through the enrichment of hub proteins of networks and as targets for posttranslational modifications. Although disordered regions are readily identified because of their distinct sequence characteristics, it is difficult to predict the functions associated with these regions. This is because disordered regions often house short (two- to five-residue) linear motifs that mediate intermolecular interactions. Predicting their function requires the ability to identify the functionally relevant motifs. If one assumes that functional motifs are highly conserved as compared to background sequence contexts, then a suitable comparative genomics approach proves to be powerful in unmasking functional motifs that are part of disordered regions. This approach has successfully identified known functional motifs and predicted a set of new motifs that might yield important insights regarding previously unknown functionalities for disordered regions. Given knowledge of highly conserved motifs, one can assess whether the rapidly changing sequence contexts are actuators of the functionalities of short linear motifs within disordered regions. This should have important implications for engineering and targeting hub proteins in signaling networks.

Hepatitis C virus NS3 helicase couples ATP binding and hydrolysis to polynucleotide unwinding. Understanding its regulation mechanism of ATP binding will facilitate targeting of ATP binding site for potential hepatitis C treatment. T324, a residue connecting domain I and domain II of NS3 helicase, has been suggested as part of a flexible hinge responsible for opening of ATP binding cleft, although the detailed mechanism remains largely unclear. We used computational simulation to examine the mutational effect of T324 on the dynamics of ATP binding site. A mutant model of T324A of NS3 helicase apo structure was created and energy minimized. Molecular dynamics simulation was conducted for both wild-type apo structure and T324A mutant to compare their differences. For the mutant structure, histogram analysis of pairwise distances between residues in domains I and II (E291-Q460, K210-R464 and R467-T212) showed that separation between the two domains was reduced bỹ 10% and the standard deviation was reduced by~33%. Principal component analysis (PCA) revealed a drastic change in the motion of first principal mode, where the mutant structure moves differently from a scissor-like opening of domains I-II of wild-type structure. RMS fluctuation (RMSF) analysis showed residues in close proximity of residue 324 (S211 and E291 in domain I as well as A458 and Q459 in domain II) have at least 30% RMSF value reductions in the mutant structure. RMSF analysis of solvent showed more water molecules are trapped near K210, S211, D290, and H293 in domain I as well as T483 and D454 in domain II to form an extensive interaction network constraining cleft opening. Our mechanistic studies revealed that an atomic interaction cascade from T324 to residues in domains I and II controls the flexibility of ATP binding cleft. 2038-Pos Board B24Multidomain Dynamics of a Fatty Acid b-Oxidation Multienzyme Complex Studied by Molecular Dynamics Simulation Tadaomi Furuta, Tohru Terada, Akinori Kidera. A fatty acid b-oxidation multienzyme complex (FOM) has been the subject of intense investigation for the elucidation of the important role in the catabolic processes for fatty acid utilization, serving as a ''hub'' in the metabolic network. The crystal structures of FOM revealed that FOM forms a heterotetrameric structure of two a and two b subunits, which covers three steps of the enzymatic reactions in the fatty acid b-oxidation. It has been proposed that the process of the multi-step reactions needs significant structure changes as observed in the large structural difference in the two crystal structures (Form I and Form II; RMSD = 4.6 Å ). To examine the flexibility of FOM, we performed 100-ns molecular dynamics simulations for the two forms without ligands (the systems contain almost 4x10 5 atoms). As the results of the simulations, we observed extremely large conformational fluctuations (RMSD R 5 Å ) in both forms. The center of mass of the a subunits (domains a M , and a C ) moved more than 10 Å against the b subunits. During the fluctuations, th...