A Simple Generic Model of Elastin-Like Polypeptides with Proline Isomerization.

A generic model of elastin-like polypeptides (ELP) is derived that includes proline isomerization (ProI). As a case study, conformational transition of a -[valine-proline-glycine-valine-glycine]- sequence is investigated in aqueous ethanol mixtures. While the non-bonded interactions are based on the Lennard-Jones (LJ) parameters, the effect of ProI is incorporated by tuning the intramolecular 3- and 4-body interactions known from the underlying all-atom simulations into the generic model. One of the key advantages of such a minimalistic model is that it readily decouples the effects of geometry and the monomer-solvent interactions due to the presence of ProI, thus gives a clearer microscopic picture that is otherwise rather nontrivial within the all-atom setups. These results are consistent with the available all-atom and experimental data. The model derived here may pave the way to investigate large scale self-assembly of ELPs or biomimetic polymers in general.

Multivalent DNA and nucleosome acidic patch interactions specify VRK1 mitotic localization and activity.

A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine-threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.

The Effects of Proline Isomerization on the Solvation Behavior of Elastin-Like Polypeptides in Water-Ethanol Mixtures.

Elastin-like polypeptides (ELPs) are well-known proline-rich stimulus-responsive polymers. They have broad applications ranging from drug delivery to green chemistry. Recently, the authors have shown that the cis/trans proline isomerization can be used to regulate their conformational behavior while keeping the lower critical solution temperature (LCST) unchanged in pure water. In aqueous ethanol mixtures, ELPs typically exhibit an expanded-collapsed-expanded transition known as the co-non-solvency phenomenon. Since it is unclear how proline isomerization affects the solvation behavior of ELPs in aqueous ethanol mixtures, an all-atom insight on single ELPs has been provided to address this question. It is found that if all proline residues are in the cis state, the peptides only experience a collapsed-expanded transition as ethanol concentration increases, i.e., the initial collapse vanishes because cis isomers prefer the compact structures in pure water. The data from the authors also suggest that proline isomerization does not change the shift in solvation free energy of an ELP with given sequence, but it varies the affinity of the peptide to both the solvent and cosolvent molecules.

Nonadditive ion effects on the coil-globule equilibrium of PNIPAM: a computer simulation study.

The combined effect of a weakly hydrated and a strongly hydrated anion on the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide)(PNIPAM) is nonadditive (Bruce et al., J. Am. Chem. Soc., 2019, 141, 6609). Herein, we revisit the molecular origin of this effect by performing atomistic molecular dynamics simulations of a 40mer PNIPAM chain in solutions with a fixed concentration of Na2SO4 and an increasing concentration of NaI. Our results show that the nonadditive ion effects on the coil-to-globule transitions of PNIPAM arise due to the interplay between the depletion of the strongly hydrated sulfate ions and the preferential accumulation of the iodide ions on the polymer surface leading to favourable PNIPAM-I- interactions. The depletion of the sulfate ions and the binding of the iodide ions are coupled through the role of the cation leading to a mutual enhancement of both effects. This mutual enhancement effect correlates with the partitioning of the Na+ cations from the counterion cloud of the weakly hydrated iodide ions to the counterion cloud of the strongly hydrated sulfate ions and the corresponding changes in water affinity of the ions.

Bufalin induces mitochondrial dysfunction and promotes apoptosis of glioma cells by regulating Annexin A2 and DRP1 protein expression.

BACKGROUND: Glioma is a common primary central nervous system tumour, and therapeutic drugs that can effectively improve the survival rate of patients in the clinic are lacking. Bufalin is effective in treating various tumours, but the mechanism by which it promotes the apoptosis of glioma cells is unclear. The aim of this study was to investigate the drug targets of bufalin in glioma cells and to clarify the apoptotic mechanism. METHODS: Cell viability and proliferation were evaluated by CCK-8 and colony formation assays. Then, the cell cycle and apoptosis, intracellular ion homeostasis, oxidative stress levels and mitochondrial damage were assessed after bufalin treatment. DARTS-PAGE technology was employed and LC-MS/MS was performed to explore the drug targets of bufalin in U251 cells. Molecular docking and western blotting were performed to identify potential targets. siRNA targeting Annexin A2 and the DRP1 protein inhibitor Mdivi-1 were used to confirm the targets of bufalin. RESULTS: Bufalin upregulated the expression of cytochrome C, cleaved caspase 3, p-Chk1 and p-p53 proteins to induce U251 cell apoptosis and cycle arrest in the S phase. Bufalin also induced oxidative stress in U251 cells, destroyed intracellular ion homeostasis, and caused mitochondrial damage. The expression of mitochondrial division-/fusion-related proteins in U251 cells was abnormal, the Annexin A2 and DRP1 proteins were translocated from the cytoplasm to mitochondria, and the MFN2 protein was released from mitochondria into the cytoplasm after bufalin treatment, disrupting the mitochondrial division/fusion balance in U251 cells. CONCLUSIONS: Our research indicated that bufalin can cause Annexin A2 and DRP1 oligomerization on the surface of mitochondria and disrupt the mitochondrial division/fusion balance to induce U251 cell apoptosis.

Tetrahydrocurcumin mitigates acute hypobaric hypoxia-induced cerebral oedema and inflammation through the NF-κB/VEGF/MMP-9 pathway.

High-altitude cerebral oedema (HACE) is a potentially fatal manifestation of high-altitude sickness and is caused partly by inflammation and the blood-brain barrier disruption. Tetrahydrocurcumin (THC) has been reported to exert effective antioxidative and anti-inflammatory effects; This study sought to elucidate the underlying mechanism of THC in mitigating HACE using a mouse model. Our results revealed that prophylactic administration of THC (40 mg/kg) for 3 days significantly alleviated the increase in brain water content (BWC), interleukin-1ß (IL-1ß) and TNF-α levels caused by acute hypobaric hypoxia (AHH). Additionally, superoxide dismutase (SOD) activity was increased by THC to enhance the ability to resist hypoxia. Histological and ultrastructural analysis of the cerebrum revealed that THC administration mitigated AHH-induced pericellular oedema and reduced the perivascular space, resulting in the simultaneous remission of oedema and protection of mitochondria in the cerebrum. In vitro, astrocytes exposed to hypoxia (4% O2 ) for 24 hr exhibited and increase in IL-1ß expression followed by an increase in vascular endothelial growth factor (VEGF) levels. Furthermore, THC administration remarkably downregulated VEGF, matrix metallopeptidase-9 (MMP-9), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, both in vivo and in vitro. Our data highlight the potential prophylactic activity of THC in HACE, it effectively mitigates AHH-induced cerebral oedema and inflammation is associated with the inhibition of the NF-κB/ VEGF/MMP-9 pathways.

Toxicity Changes of Heavily Polluted River Sediments on Daphnia magna Before and After Dredging.

Most of the pollutants discharged into the water will deposit at the bottom of the river and may cause biological toxicity. Daphnia magna-elutriate toxicity bioassay was usually applied to evaluate sediment toxicity. However, the loss of hydrophobic pollutants during the elutriating will lead to the underestimation of sediment toxicity. The purpose of this study is to apply the optimized immobilized sediments to D. magna test, so it can be directly exposed to the sediments and get accurate sediment toxicity results. The optimized immobilized sediment was prepared by mixing 1 g sediment with 7.5 mL 3% (w/v) alginate and hardened in a 4% (w/v) CaCl2 solution. Based on D. magna acute toxicity test, the median lethal concentration values (LC50) of the spiked Cu and diuron measured by using immobilized sediment were both lower than that of using the elutriate, in which the difference of Cu-LC50 reached a significant level. The toxicity changes of sediment in the polluted rivers before and after dredging were then be evaluated by using the immobilized sediment. The toxicity of the sediments at four sites decreased from acute-toxic (pro-dredging) to slight-acute-toxic and nontoxic (post-dredging).

The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans.

Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22 SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing.

The exclusive effects of chaperonin on the behavior of proteins with 52 knot.

The folding of proteins with a complex knot is still an unresolved question. Based on representative members of Ubiquitin C-terminal Hydrolases (UCHs) that contain the 52 knot in the native state, we explain how UCHs are able to unfold and refold in vitro reversibly within the structure-based model. In particular, we identify two, topologically different folding/unfolding pathways and corroborate our results with experiment, recreating the chevron plot. We show that confinement effect of chaperonin or weak crowding greatly facilitates folding, simultaneously slowing down the unfolding process of UCHs, compared with bulk conditions. Finally, we analyze the existence of knots in the denaturated state of UCHs. The results of the work show that the crowded environment of the cell should have a positive effect on the kinetics of complex knotted proteins, especially when proteins with deeper knots are found in this family.

Stability of structurally entangled protein dimers.

We studied stretching, folding and thermodynamic properties of structurally entangled protein dimers. The tests for entanglement involve four-terminal pulling. We study the dynamics of such pulling and contrast it with the standard two-terminal one. The two-chain entanglement is qualitatively characterized by its entangled core, which is defined as the minimal structure that is entangled. The existence of the entangled cores is found to be affecting both the mechanical and folding properties of the proteins. We also show that the folding pathways of the entangled proteins are not universal but the bottleneck is always the formation of the entangled conformation. We demonstrate that entanglement enhances thermodynamic stability.

Structural Changes in Barley Protein LTP1 Isoforms at Air-Water Interfaces.

We use a coarse-grained model to study the conformational changes in two barley proteins, LTP1 and its ligand adduct isoform LTP1b, that result from their adsorption to the air-water interface. The model introduces the interface through hydropathy indices. We justify the model by all-atom simulations. The choice of the proteins is motivated by making attempts to understand formation and stability of foam in beer. We demonstrate that both proteins flatten out at the interface and can make a continuous stabilizing and denser film. We show that the degree of the flattening depends on the protein (the layers of LTP1b should be denser than those of LTP1) and on the presence of glycation. It also depends on the number (≤4) of the disulfide bonds in the proteins. The geometry of the proteins is sensitive to the specificity of the absent bonds. We provide estimates of the volume of cavities of the proteins when away from the interface.

Proteins at air-water and oil-water interfaces in an all-atom model.

We study the behavior of five proteins at the air-water and oil-water interfaces by all-atom molecular dynamics. The proteins are found to get distorted when pinned to the interface. This behavior is consistent with the phenomenological way of introducing the interfaces in a coarse-grained model through a force that depends on the hydropathy indices of the residues. Proteins couple to the oil-water interface stronger than to the air-water one. They diffuse slower at the oil-water interface but do not depin from it, whereas depinning events are observed at the other interface. The reduction of the disulfide bonds slows the diffusion down.

Structural entanglements in protein complexes.

We consider multi-chain protein native structures and propose a criterion that determines whether two chains in the system are entangled or not. The criterion is based on the behavior observed by pulling at both termini of each chain simultaneously in the two chains. We have identified about 900 entangled systems in the Protein Data Bank and provided a more detailed analysis for several of them. We argue that entanglement enhances the thermodynamic stability of the system but it may have other functions: burying the hydrophobic residues at the interface and increasing the DNA or RNA binding area. We also study the folding and stretching properties of the knotted dimeric proteins MJ0366, YibK, and bacteriophytochrome. These proteins have been studied theoretically in their monomeric versions so far. The dimers are seen to separate on stretching through the tensile mechanism and the characteristic unraveling force depends on the pulling direction.

Plants affect the horizontal transmission of a new densovirus infecting the green peach aphid Myzus persicae by modulating honeydew production.

In a tritrophic context of plant-insect-entomopathogen, plants play important roles in modulating the interaction of insects and their pathogenic viruses. Currently, the influence of plants on the transmission of insect viruses has been mainly studied on baculoviruses and some RNA viruses, whereas the impact of plants on other insect viruses is largely unknown. Here, we identified a new densovirus infecting the green peach aphid Myzus persicae and tested whether and how host plants influence the transmission of the aphid densovirus. The complete single-stranded DNA genome of the virus, M. persicae densovirus 2, is 5 727 nt and contains inverted terminal repeats. Transcription and phylogenetic analysis indicated that the virus was distinct from other a few identified aphid densoviruses. The virus abundance was detected highly in the intestinal tract of aphids, compared with the lower level of it in other tissues including head, embryo, and epidermis. Cabbage and pepper plants had no obvious effect on the vertical transmission and saliva-mediated horizontal transmission of the virus. However, the honeydew-mediated horizontal transmission among aphids highly depended on host plants (65% on cabbages versus 17% on peppers). Although the virus concentration in the honeydew produced by aphids between 2 plants was similar, the honeydew production of the infected aphids reared on peppers was dramatically reduced. Taken together, our results provide evidence that plants influence the horizontal transmission of a new densovirus in an aphid population by modulating honeydew secretion of aphids, suggesting plants may manipulate the spread of an aphid-pathogenic densovirus in nature.

Rapid vision improvement by using icotinib in a patient with bilateral choroidal metastases symmetrically from lung cancer.

INTRODUCTION: The metastases of lung cancer to bilateral choroids symmetrically and simultaneously are very rare. Almost all patients with choroid metastasis can be treated with external beam radiotherapy in order to increase quality of life and preserve vision. MATERIAL AND METHODS: We documented a case and studied the effect of icotinib on choroidal metastases in bilateral eyes simultaneously from pulmonary adenocarcinoma. RESULTS: A 49-year-old Chinese man presented with bilateral vision losing simultaneously for 4 weeks, it was as an initial presentation in the clinical. The examinations with ophthalmofundoscopy, ultrasonography, and fluorescein angiography showed the lesions in bilateral choroids, two solitary juxtapapillary yellow-white choroidal metastases inferior to the optic discs with bleeding. Positron emission tomography confirmed the choroidal metastases and further proved that it was from lung cancer with lymph nodes and multiple bone metastasis. The biopsy taken from the lung by bronchoscopy and needle biopsy from supraclavicular lymph nodes revealed the pulmonary adenocarcinoma with epithelial growth factor receptor mutation (exon 21). The patient was treated with oral icotinib (125 mg, three times a day, TID). Five days after starting icotinib therapy, the patient's visions were rapidly recovered. Two months after the treatment with icotinib, the choroidal metastases regressed to small lesions, and the visions were preserved to before. The lung tumor and other metastatic lesions were partly regressive. There was no evidence of recurrence for eye lesions at 15-months follow-up. After 17 months treating by icotinib, the patient presented headache and dizzy with multiple brain metastases determined by magnetic resonance imaging; however, the lesions of the choroidal metastases remained progressing-free. Almonertinib with radiotherapy were used to treat the brain metastases, and he is surviving with progress-free more than 2 years until now. CONCLUSION: Bilateral choroidal metastases from lung cancer symmetrically are very rare. Icotinib following by almonertinib was an alternative therapy for choroidal metastasis from non-small cell lung cancer with epithelial growth factor receptor mutation.

Stabilizing α-Helicity of a Polypeptide in Aqueous Urea: Dipole Orientation or Hydrogen Bonding?

We propose a mechanism for α-helix folding of polyalanine in aqueous urea that reconciles experimental and simulation studies. Over 15 µs long, all-atom simulations reveal that, upon dehydrating the protein's first solvation shell, a delicate balance between localized urea-residue dipole interactions and hydrogen bonds dictates polypeptide solvation properties and structure. Our work clarifies the experimentally observed tendency of these alanine-rich systems to form secondary structures at low and intermediate urea concentrations. Moreover, it is consistent with the commonly accepted hydrogen-bond-induced helix unfolding, dominant at high urea concentrations. These results establish a structure-property relationship highlighting the importance of microscopic dipole-dipole orientations/interactions for the operational understanding of macroscopic protein solvation.

Can Polymer Helicity Affect Topological Chirality of Polymer Knots?

We investigate the effect of helicity in isolated polymers on the topological chirality of their knots with computer simulations. Polymers are described by generic worm-like chains (WLC), where helical conformations are promoted by chiral coupling between segments that are neighbors along the chain contour. The sign and magnitude of the coupling coefficient u determine the sense and strength of helicity. The corrugation of the helix is adjusted via the radius R of a spherical, hard excluded volume around each WLC segment. Open and compact helices are, respectively, obtained for R that is either zero or smaller than the length of the WLC bond, and R that is a few times larger than the bond length. We use a Monte Carlo algorithm to sample polymer conformations for different values of u, spanning the range from achiral polymers to chains with well-developed helices. Monitoring the average helix torsion and fluctuations of chiral order as a function of u, for two very different chain lengths, demonstrates that the coil-helix transition in this model is not a phase transition but a crossover. Statistical analysis of conformations forming the simplest chiral knots, 31, 51, and 52, demonstrates that topological mirror symmetry is broken─knots formed by helices with a given sense prefer one handedness over the other. For the 31 and 51 knots, positive helical sense favors positive handedness. Intriguingly, an opposite trend is observed for 52 knots, where positive helical sense promotes negative handedness. We argue that this special coupling between helicity and topological chirality stems from a generic mechanism: conformations where some of the knot crossings are found in "braids" formed by two tightly interwoven sections of the polymer.

Paeonia × suffruticosa Andrews leaf extract and its main component apigenin 7-O-glucoside ameliorate hyperuricemia by inhibiting xanthine oxidase activity and regulating renal urate transporters.

BACKGROUND: Hyperuricemia is an important pathological basis of gout and a distinct hazard factor for metabolic syndromes and cardiovascular and chronic renal disease, but lacks safe and effective treatments currently. Paeonia × suffruticosa Andrews leaf effectively reduced serum uric acid in gout patients; however, the material foundation and the mechanism remain unclear. PURPOSE: To determine the primary active components and mechanism of P. suffruticosa leaf in hyperuricemic mice. METHODS: The chemical constituents of P. suffruticosa leaf was identified using high-performance liquid chromatographic analysis. The anti-hyperuricemic activity of P. suffruticosa leaf extract (12.5, 25, 50, 100, and 200 mg/kg) and its components was evaluated in hyperuricemic mice induced by a high purine diet for 14 days. Then, the urate-lowering effects of apigenin 7-O-glucoside (0.09, 0.18, and 0.36 mg/kg) were assessed in another hyperuricemic mice model built by administrating potassium oxonate and adenine for 4 weeks. The inhibitory effect of apigenin 7-O-glucoside on uric acid production was elucidated by investigating xanthine oxidase activity in vitro and in serum and the liver and through molecular docking. Immunofluorescence and western blot analyses of the expression of renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporters 1 (OAT1), and ATP-binding cassette G member 2 (ABCG2) proteins elucidated how apigenin 7-O-glucoside promoted uric acid excretion. RESULTS: Six compounds were identified in P. suffruticosa leaf: gallic acid, methyl gallate, oxypaeoniflorin, paeoniflorin, galloylpaeoniflorin, and apigenin 7-O-glucoside. P. suffruticosa leaf extract significantly attenuated increased serum uric acid, creatinine, and xanthine oxidase activity in hyperuricemic mice. Apigenin 7-O-glucoside from P. suffruticosa leaf reduced uric acid, creatinine, and malondialdehyde serum levels, increased superoxide dismutase activity, and partially restored the spleen coefficient in hyperuricemic mice. Apigenin 7-O-glucoside inhibited xanthine oxidase activity in vitro and decreased serum and liver xanthine oxidase activity and liver xanthine oxidase protein expression in hyperuricemic mice. Molecular docking revealed that apigenin 7-O-glucoside bound to xanthine oxidase. Apigenin 7-O-glucoside facilitated uric acid excretion by modulating the renal urate transporters URAT1, GLUT9, OAT1, and ABCG2. Apigenin 7-O-glucoside protected against renal damage and oxidative stress caused by hyperuricemia by reducing serum creatinine, blood urea nitrogen, malondialdehyde, and renal reactive oxygen species levels; increasing serum and renal superoxide dismutase activity; restoring the renal coefficient; and reducing renal pathological injury. CONCLUSION: Apigenin 7-O-glucoside is the main urate-lowering active component of P. suffruticosa leaf extract in the hyperuricemic mice. It suppressed liver xanthine oxidase activity to decrease uric acid synthesis and modulated renal urate transporters to stimulate uric acid excretion, alleviating kidney damage caused by hyperuricemia.

Combined effects of dietary carbohydrate levels and ammonia stress on growth, antioxidant capacity and glucose metabolism in juvenile oriental river prawn (Macrobrachium nipponense).

Ammonia is a common environmental stress factor that constrains aquaculture industry development. This study evaluated the effect of carbohydrate levels and ammonia stress in oriental river prawn (Macrobrachium nipponense). The experiment had six treatments containing two water ammonia levels (0 and 5 mg/L) and three dietary carbohydrate levels (low carbohydrate diet (LCD, 10%), medium carbohydrate diet [MCD, 20%], and high carbohydrate diet [HCD, 30%]), and lasted six weeks. The results showed that the prawns fed on MCD had higher weight gain than those fed on LCD and HCD during ammonia stress. Moreover, the prawns fed on MCD had significantly lower acid phosphatase and alkaline phosphatase activities during ammonia stress. Feeding the prawns on the MCD increased B cells in the hepatopancreas during ammonia stress. Interestingly, the prawns fed on MCD had significantly lower superoxide dismutase activity compared to LCD and HCD during ammonia stress. Moreover, the prawns fed on MCD had significantly lower pyruvate kinase activity and pyruvate and lactic acid contents, while those fed on LCD had significantly higher succinic dehydrogenase, 6-phosphogluconic dehydrogenase, and phosphoenol pyruvate carboxykinase activities during ammonia stress. The prawns fed on the MCD increased significantly glutaminase activity and decreased the ammonia content in the serum during ammonia exposure. In addition, feeding the prawns on MCD decreased significantly the expression of apoptosis and inflammation-related genes. Taken together, the MCD supplied energy required to counteract ammonia stress, which increased growth, improved antioxidant capacity, facilitated ammonia excretion, and alleviated inflammation and apoptosis of the oriental river prawn.

Solvation Behavior of Elastin-like Polypeptides in Divalent Metal Salt Solutions.

The effects of CaCl2 and MgCl2 on the cloud point temperature of two different elastin-like polypeptides (ELPs) were studied using a combination of cloud point measurements, molecular dynamics simulations, and infrared spectroscopy. Changes in the cloud point for the ELPs in aqueous divalent metal cation solutions were primarily governed by two competing interactions: the cation-amide oxygen electrostatic interaction and the hydration of the cation. In particular, Ca2+ cations can more readily shed their hydration shells and directly contact two amide oxygens by the formation of ion bridges. By contrast, Mg2+ cations were more strongly hydrated and preferred to partition toward the amide oxygens along with their hydration shells. In fact, although hydrophilic ELP V5A2G3 was salted-out at low concentrations of MgCl2, it was salted-in at higher salt concentrations. By contrast, CaCl2 salted the ELP sharply out of solution at higher salt concentrations because of the bridging effect.