Competing interactions give rise to two-state behavior and switch-like transitions in charge-rich intrinsically disordered proteins

The most commonly occurring intrinsically disordered proteins (IDPs) are polyampholytes, which are defined by the duality of low net charge per residue and high fractions of charged residues. Recent experiments have uncovered nuances regarding sequence–ensemble relationships of model polyampholytic IDPs. These include differences in conformational preferences for sequences with lysine vs. arginine and the suggestion that well-mixed sequences form a range of conformations, including globules, conformations with ensemble averages that are reminiscent of ideal chains, or self-avoiding walks. Here, we explain these observations by analyzing results from atomistic simulations. We find that polyampholytic IDPs generally sample two distinct stable states, namely, globules and self-avoiding walks. Globules are favored by electrostatic attractions between oppositely charged residues, whereas self-avoiding walks are favored by favorable free energies of hydration of charged residues. We find sequence-specific temperatures of bistability at which globules and self-avoiding walks can coexist. At these temperatures, ensemble averages over coexisting states give rise to statistics that resemble ideal chains without there being an actual counterbalancing of intrachain and chain-solvent interactions. At equivalent temperatures, arginine-rich sequences tilt the preference toward globular conformations whereas lysine-rich sequences tilt the preference toward self-avoiding walks. We also identify differences between aspartate- and glutamate-containing sequences, whereby the shorter aspartate side chain engenders preferences for metastable, necklace-like conformations. Finally, although segregation of oppositely charged residues within the linear sequence maintains the overall two-state behavior, compact states are highly favored by such systems.

Significant fractions of eukaryotic proteomes are made up of intrinsically disordered regions (IDRs) (1). Conformational heterogeneity (2) is a defining hallmark of IDRs (3–5). Studies over the past decade have helped quantify relationships (6) that connect sequence-encoded information within IDRs to properties of conformational ensembles such as overall sizes and shapes, the amplitudes of spontaneous conformational fluctuations, and the dynamics of interconverting between distinct conformational states (7–19). These sequence–ensemble relationships have direct functional consequences that have been uncovered via studies based on biophysical, biochemical, and engineering approaches (5, 20–38). Our work, which is focused on physical principles underlying sequence–ensemble relationships of IDRs, is of direct relevance to understanding how IDRs function.Charged residues are key determinants of sequence–ensemble relationships of IDRs (19, 39–41). They contribute through highly favorable free energies of hydration (42) and long-range electrostatic interactions. Net charge per residue (19, 39, 40) and the patterning of oppositely charged residues (43–45) are useful order parameters for describing sequence–ensemble relationships and interactions of charge-rich IDRs (46). Both features can be modulated through posttranslational modifications (47–51), charge renormalization by solution ions (52), and charge regulation through context- and conformation-dependent uptake and release of protons (53).Polyampholytes feature roughly equivalent numbers of oppositely charged residues, and they make up more than 70% of known IDRs (7, 17). For a given amino acid composition, which sets the fraction of charged residues and the net charge per residue, it has been shown that the linear mixing vs. segregation of oppositely charged residues can have a profound impact on sequence–ensemble relationships of polyampholytic IDRs (31, 32, 43). Specifically, for a given set of solution conditions, sequences featuring uniform linear distributions of oppositely charged residues are predicted to favor more expanded conformations compared to sequences with identical amino acid compositions where the oppositely charged residues are segregated into distinct blocks along the linear sequence. These predictions made using simulation and theory (43, 44, 54) have been confirmed using different experiments (31–33, 41, 55).The ensemble-averaged radii of gyration (R_g) of flexible polymers follow scaling relationships of the form R_g ∼ N^ν. Here, N denotes the number of residues and the scaling exponent ν is a measure of the length scale over which conformational fluctuations are correlated. For homopolymers or systems that are effective homopolymers, ν has four limiting values, viz., 0.33, 0.5, 0.59, or 1, corresponding to globules, Flory random coils (FRCs), self-avoiding walks, and rod-like conformations, respectively (56). Atomistic simulations performed at fixed temperatures suggest that ν ≈ 0.59 (43) for strong, well-mixed polyampholytes (17). The explanation for this behavior is as follows. Electrostatic attractions and repulsions are realized on similar length scales for well-mixed sequences. These interactions screen one another, and the highly favorable free energies of hydration become the main determinants of overall sizes and shapes of well-mixed strong polyampholytes (17). In contrast, compact conformations are formed by strong polyampholytes where oppositely charged residues are segregated into distinct blocks. Here, the electrostatic attractions between oppositely charged blocks can outcompete opposing effects of favorable solvation. These inferences were gleaned using sequences comprising 1:1 ratios of Lys and Glu (43). In the original simulations, the reference free energies of hydration of all charged residues were treated as being quantitatively equivalent and highly favorable. This leads to the hypothesis that Lys and Arg are interoperable with one another as determinants of sequence–ensemble relationships of IDRs (17). A similar inference emerges regarding the interoperability of Asp and Glu with respect to one another. The recent work of Sørensen and Kjaergaard has challenged these inferences (57). Using a system where model IDRs were deployed as flexible linkers between interaction domains, Sørensen and Kjaergaard used their measurements to estimate the relationships between amino acid sequence and the scaling exponent ν (57). Inferences from their experiments suggest that the ν ≈ 0.33 for (GRESRE)_n and ν ≈ 0.5 for (GKESKE)_n for the specific conditions they used in their measurements. Here, n is the number of repeats of the hexapeptides GRESRE or GKESKE. The results point to significant differences between Arg- and Lys-containing sequences. Further, while globularity of (GRESRE)_n has precedent in mean-field theories for polyampholytes, the mechanism by which FRC-like behavior of (GKESKE)_n is achieved is unclear. Here, we develop a plausible physical explanation for the findings of Sørensen and Kjaergaard (57). Our work is based on atomistic simulations and the ABSINTH implicit solvation model and forcefield paradigm (58–61).     

Net charge per residue modulates conformational ensembles of intrinsically disordered proteins

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.     

不对称树状聚两性电解质的合成及pH响应性自组装行为

通过发散法、开环聚合以及“点击化学”等方法合成了树状聚两性电解质聚（L-赖氨酸）-聚酰胺-胺-聚（L-谷氨酸）[（PLL）₄-D2-b-D1-（PLGA）₂],其中,D1和D2分别代表第1代和第2代聚酰胺-胺。通过氢核磁共振波谱（¹H-NMR）、傅里叶变换红外光谱（FT-IR）和凝胶渗透色谱（GPC）对产物的分子结构、相对分子质量及其分布进行了表征;结合电位电导滴定、¹H-NMR、Zeta电位测定、扫描电子显微镜（SEM）、透射电子显微镜（TEM）和动态光散射（DLS）等表征方法研究了（PLL）₄-D2-b-D1-（PLGA）₂溶液的pH响应性自组装行为。研究结果表明,（PLL）₄-D2-b-D1-（PLGA）₂具有pH响应性多重胶束化行为,在酸性条件下形成PLL为壳、PLGA为核的聚集体,而在碱性条件下形成PLGA为壳、PLL为核的聚集体,同时伴随着PLGA和PLL链段从无规线团到α-螺旋构象的转变。随着溶液pH的增加,自组装聚集体由球形胶束向棒状胶束再向纺锤形胶束转变,并在高pH下二次自组装形成松叶状分形结构。     

Cryoprotective properties of completely synthetic polyampholytes via reversible addition-fragmentation chain transfer (RAFT) polymerization and the effects of hydrophobicity

A completely synthetic polyampholyte cryoprotectant was developed with cationic and anionic monomers by reversible addition-fragmentation chain transfer polymerization. The neutralized random polyampholyte, which had an equal composition ratio of monomers, showed high cryoprotective properties in mammalian cells. Introduction of a small amount of hydrophobic monomer enhanced cell viability after cryopreservation, indicating the importance of hydrophobicity. Leakage experiments confirmed that these polyampholytes protected the cell membrane during cryopreservation. Due to low cytotoxicity, this polyampholyte has the potential to replace the convention cryoprotective agent dimethyl sulfoxide. The present study is the first to show that we can design a polymeric cryoprotectant that will protect the cell membrane during freezing using appropriate polymerization techniques.     

铆接于固体表面的两性聚电解质链构象的分子动力学模拟

用分子动力学模拟研究了铆接于带电固体壁面上的两性无规共聚电解质链的构型及其随溶剂的介电常数、系统温度和带电壁面电场的变化。结果表明，聚离子链的构型取决于外电场对链节的作用、链内各链节间的静电作用和链节的热运动(温度)三者的综合作用结果。电场强度较低时，铆接聚离子链与非铆接聚离子链的构型变化类似。电场强度较高时，两性聚离子链构型更为舒展。溶剂介电常数减小，链节间静电作用增强，净电荷比较小的中性或非中性两性聚电解质链倾向于收缩，净电荷比较多的链则膨胀。温度的升高一般总是抵消电场和分子内静电作用的影响，当它占主导地位时将使聚离子链膨胀。     

两性聚电解质PDMAm-b-PAAn与表面活性剂（12-6-12）的相互作用

采用Zeta电位、荧光探针、表面张力和黏度等方法研究了碱性条件下不同嵌段比的两性聚电解质聚(N,N-二甲胺基甲基丙烯酸乙酯-b-丙烯酸)(PDMAm-b-PAAn)与阳离子偶联表面活性剂（C12H25(CH3)2N(CH2)6N(CH3)2C12H25·2Br-) (简称12-6-12)的相互作用。结果表明：由于静电相互作用,两嵌段聚电解质PDMAm-b-PAAn和12-6-12之间可形成类胶束或复合物,PDMA链段的弱亲水性对复合物起到稳定的作用。对同一类型的两嵌段聚电解质,改变两链段的相对长度之比,既不会使其在溶液中的构象发生改变,也不会使其与表面活性剂的相互作用模式发生改变。     

From the Cover: Charge interactions can dominate the dimensions of intrinsically disordered proteins

Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically disordered proteins (IDPs) often contain a large fraction of charged amino acids. Here, we use single-molecule Förster resonance energy transfer to investigate the influence of charged residues on the dimensions of unfolded and intrinsically disordered proteins. We find that, in contrast to the compact unfolded conformations that have been observed for many proteins at low denaturant concentration, IDPs can exhibit a prominent expansion at low ionic strength that correlates with their net charge. Charge-balanced polypeptides, however, can exhibit an additional collapse at low ionic strength, as predicted by polyampholyte theory from the attraction between opposite charges in the chain. The pronounced effect of charges on the dimensions of unfolded proteins has important implications for the cellular functions of IDPs.     

Long-term cryopreservation of human mesenchymal stem cells using carboxylated poly-l-lysine without the addition of proteins or dimethyl sulfoxide

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are known for their potential to undergo mesodermal differentiation into many cell types, including osteocytes, adipocytes, and chondrocytes. Therefore, hBMSCs can be used for a variety of regenerative medicine therapies, in fact, hBMC-derived osteocytes have already been used in bone reconstruction. This study discusses the viability and the differentiation properties of hBMSCs that have been cryopreserved in the absence of proteins or dimethyl sulfoxide (DMSO) by using a novel polyampholyte cryoprotective agent (CPA). This CPA is based on carboxylated poly-l-lysine (COOH-PLL) and it was produced by a reaction between ?-poly-l-lysine and succinic anhydride. ¹H-NMR and two-dimensional correlation (¹H-¹³C HSQC) spectroscopy revealed that COOH-PLL did not have a special structure in solution. The hBMSCs can be cryopreserved for 24?months at ?80?°C by using a 7.5% (w/w) cryopreserving solution of COOH-PLL, which introduces carboxyl groups that result in?>?90% cell viability after thawing. Furthermore, the cryopreserved hBMSCs fully retained both their proliferative capacity as well as their potential for osteogenic, adipogenic, and chondrogenic differentiation. Confocal laser-scanning microscopy showed that the polyampholyte CPA did not penetrate the cell membrane; rather, it attached to the membrane during cryopreservation. These results indicate that the cryoprotective mechanisms of COOH-PLL might differ from those of currently used small molecule CPAs. These results also suggest that using COOH-PLL as a CPA for hBMSC preservation can eliminate the use of proteins and DMSO, which would be safer if these cells were used for cell transplantation or regenerative medicine.     

两性高分子的溶液性质

两性高分子是分子链上同时含有正负电荷基团的一类高聚物、具有独特的溶液性质。本文就两性高分子等电点的构象状态、影响等电点的因素、两性高分子的溶解性以及高分子结构和组成、ｐＨ、溶液离子强度、混合溶剂、温度对流体力学行为的影响进行了综述。