Cataract-related variant R114C increases ßA3-crystallin susceptibility to environmental stresses by disrupting the protein senior structure.

Congenital cataract is a major cause of childhood blindness worldwide, with crystallin mutations accounting for over 40 % of gene-mutation-related cases. Our research focused on a novel R114C mutation in a Chinese family, resulting in bilateral coronary cataract with blue punctate opacity. Spectroscopic experiments revealed that ßA3-R114C significantly altered the senior structure, exhibiting aggregation, and reduced solubility at physiological temperature. The mutant also displayed decreased resistance and stability under environmental stresses such as UV irradiation, oxidative stress, and heat. Further, cellular models confirmed its heightened sensitivity to environmental stresses. These data suggest that the R114C mutation impairs the hydrogen bond network and structural stability of ßA3-crystallin, particularly at the boundary of the second Greek-key motif. This study revealed the pathological mechanism of ßA3-R114C and may help in the development of potential treatment strategies for related cataracts.

Subacute-onset cataract in a 29-year-old man with mitochondrial encephalomyopathy: A case report.

This case report aims to emphasize that subacute occurrence of nuclear cataract might be one of the underestimated manifestations of mitochondrial encephalomyopathy, thus periodical ophthalmologic examinations are recommended.

Electroacupuncture Relieves HuR/KLF9-Mediated Inflammation to Enhance Neurological Repair after Spinal Cord Injury.

Electroacupuncture (EA) is widely applied in clinical therapy for spinal cord injury (SCI). However, the associated molecular mechanism has yet to be elucidated. The current study aimed to investigate the underlying mechanism of EA in neurologic repair after SCI. First, we investigated the role of EA in the neurologic repair of the SCI rat model. The expression levels of human antigen R (HuR) and Krüppel-like factor 9 (KLF9) in spinal cord tissues were quantified after treatment. Second, we conducted bioinformatics analysis, RNA pull-down assays, RNA immunoprecipitation, and luciferase reporter gene assay to verify the binding of HuR and KLF9 mRNA for mRNA stability. Last, HuR inhibitor CMLD-2 was used to verify the enhanced effect of EA on neurologic repair after SCI via the HuR/KLF9 axis. Our data provided convincing evidence that EA facilitated the recovery of neuronal function in SCI rats by reducing apoptosis and inflammation of neurons. We found that EA significantly diminished the SCI-mediated upregulation of HuR, and HuR could bind to the 3' untranslated region of KLF9 mRNA to protect its decay. In addition, a series of in vivo experiments confirmed that CMLD-2 administration increased EA-mediated pain thresholds and motor function in SCI rats. Collectively, the present study showed that EA improved pain thresholds and motor function in SCI rats via impairment of HuR-mediated KLF9 mRNA stabilization, thus providing a better understanding of the regulatory mechanisms regarding EA-mediated neurologic repair after SCI.

Cataract-causing mutations S78F and S78P of γD-crystallin decrease protein conformational stability and drive aggregation.

Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation.

The Facile Three-Dimensional Printing of the Composite of Copper Nanosized Powder and Micron Powder with Enhanced Properties.

Three-dimensional (3D) printing of Cu items is a new way to build up the structured Cu materials, but 3D printing of Cu items is usually a challenge because of the high melting point, high thermal conductivity, and high light reflection rate of Cu material. In this study, the composite of Cu microspheres powder and Cu nanoparticles (micro/nano Cu powder) is used to realize the 3D printing of Cu items with the selective laser melting technology. The sintering temperature and the thermal conductivity of micro/nano Cu powder are evidently decreased due to Cu nanoparticles' addition in the micron Cu powder. The results reveal that the 3D printing of 50%/50% micro/nano Cu powder needs laser power range of 100-240 W, which is in contrast to 200-340 W for 3D printing of 100% Cu microspheres powder. Furthermore, the conductivity, mechanical strength, and density of 3D-printed Cu items are improved with the addition of Cu nanoparticles into the micron Cu powder. The increasement of 34% on electrical conductivity and 17% on tensile strength are reached by the addition of 50% Cu nanoparticles with the laser power of 240 W.

The 18th amino acid glycine plays an essential role in maintaining the structural stabilities of γS-crystallin linking with congenital cataract.

γS-crystallin is particularly rich in the embryonic nuclear region and is crucial to the maintenance of lens transparency and optical properties. Gene mutations in crystallin are the main factors leading to congenital hereditary cataracts, which are a major cause of visual impairment in children. Some mutations located in the 18th amino acid glycine of γS-crystallin were reported to be linking with congenital cataracts. However, the pathogenic mechanism has not been elucidated. Interestingly, we previously identified a novel variant of γS-crystallin (c.53G > A; p. G18D) with progressive cortical and sutural congenital cataracts in one Chinese family. In this study, we purified the γS-crystallin wildtype and mutant proteins to investigate the effects of the G18D mutation on the structural stability of γS-crystallin. The results showed that there were tertiary structural differences between the wild-type γS-crystallin and the G18D variant. The mutation significantly impaired the stability of γS-crystallin under environmental stress and promoted aggregation. Furthermore, molecular dynamics (MD) simulations showed that the mutation altered H-bonding and surface electrostatic potential. Significantly decreased stability along with an increased tendency to aggregate under environmental stress may be the major pathogenic factors for cataracts induced by the G18D mutation.

Lens regeneration in situ using hESCs-derived cells -similar to natural lens.

Lens itself has limited regeneration functionality, thus we aimed to create regenerated lens with biological function to treat cataracts rather than the intraocular lens used in cataract surgery. We induced exogenous human embryonic stem cells to directionally differentiate into lens fate like cells in vitro, mixed these cells with hyaluronate, and then implanted the mixture into lens capsule to regenerate in vivo. We successfully achieved near-complete lens regeneration, and the thickness of the regenerated lens reached 85% of the contralateral eye, showing the characteristics of biconvex shape, transparency, and a thickness and diopter close to that of natural lenses. Meanwhile, the participation of Wnt/PCP pathway in lens regeneration was verified. The regenerated lens in this study was the most transparent, thickest, and most similar to the original natural lens that has thus far been reported. Overall, these findings offer a new therapeutic strategy for cataracts and other lens diseases.

Insight into Pathogenic Mechanism Underlying the Hereditary Cataract Caused by ßB2-G149V Mutation.

Congenital cataracts account for approximately 5-20% of childhood blindness worldwide and 22-30% of childhood blindness in developing countries. Genetic disorders are the primary cause of congenital cataracts. In this work, we investigated the underlying molecular mechanism of G149V point missense mutation in ßB2-crystallin, which was first identified in a three-generation Chinese family with two affected members diagnosed with congenital cataracts. Spectroscopic experiments were performed to determine the structural differences between the wild type (WT) and the G149V mutant of ßB2-crystallin. The results showed that the G149V mutation significantly changed the secondary and tertiary structure of ßB2-crystallin. The polarity of the tryptophan microenvironment and the hydrophobicity of the mutant protein increased. The G149V mutation made the protein structure loose and the interaction between oligomers was reduced, which decreased the stability of the protein. Furthermore, we compared ßB2-crystallin WT and the G149V mutant with their biophysical properties under environmental stress. We found that the G149V mutation makes ßB2-crystallin more sensitive to environmental stresses (oxidative stress, UV irradiation, and heat shock) and more likely to aggregate and form precipitation. These features might be important to the pathogenesis of ßB2-crystallin G149V mutant related to congenital cataracts.

Comparison of femtosecond laser-assisted cataract surgery and conventional phacoemulsification on corneal impact: A meta-analysis and systematic review.

This meta-analysis aims to compare corneal injuries and function after femtosecond laser-assisted cataract surgery (FLACS) and conventional phacoemulsification surgery (CPS). A comprehensive literature search of PubMed, EMBASE, and the Cochrane Controlled Trials Register was conducted to identify randomized controlled trials (RCT) and high-quality prospective comparative cohort studies comparing FLACS with CPS. Endothelial cell loss percentage (ECL%), central corneal thickness (CCT), endothelial cell density (ECD), endothelial cell loss (ECL), percentage of the hexagonal cell (6A), and coefficient of variance (CoV) were used as an indicator of corneal injury and function. Totally 42 trials (23 RCTs and 19 prospective cohort studies), including 3916 eyes, underwent FLACS, and a total of 3736 eyes underwent CPS. ECL% is significantly lower in the FLACS group at 1-3 days (P = 0.005), 1 week (P = 0.004), 1 month (P<0.0001), 3 months (P = 0.001), and 6 months (P = 0.004) after surgery compared to CPS. ECD and ECL appeared no statistically significant difference between the two groups, except for the significant reduction of ECD at 3 months in the CPS group (P = 0.002). CCT was significantly lower in the FLACS group at 1 week (P = 0.05) and 1 month (P = 0.002) early postoperatively. While at 1-3 days (P = 0.50), 3 months (P = 0.18), and 6 months (P = 0.11), there was no difference between the FLACS group and the CPS group. No significant difference was found in the percentage of hexagonal cells and the coefficient of variance. FLACS, compared with CPS, reduces corneal injury in the early postoperative period. Corneal edema recovered faster in the FLACS group in the early postoperative period. In addition, FLACS may be a better option for patients with corneal dysfunction.

Heteromeric formation with ßA3 protects the low thermal stability of ßB1-L116P.

BACKGROUND/AIMS: Congenital cataract is the leading cause of visual disability and blindness in childhood. ßB1-crystallin (CRYBB1) comprises about 1/10th of crystallin structural proteins, forming heteromers to maintain lens transparency. We previously reported a CRYBB1 mutation (c.347T>C, p.L116P) affecting 16 patients in a congenital nuclear cataract family. In this study, we investigate the underlying pathogenic mechanism of ßB1-L116P. METHODS: Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess ßA3- and ßB1-crystallin thermal stability, structural properties and heteromer formation. RESULTS: Cells that overexpressed ßB1-L116P tended to form aggregates and precipitations under heat-shock stress. Thermal denaturation and time-dependent turbidity experiments showed that thermal stability was significantly impaired. Moreover, protein instability appeared to increase with elevated concentrations detected by the Uncle system. Additionally, ßA3 had a relative protective effect on ßB1-L116P after heteromers were formed, although ßA3 was relatively unstable and was usually protected by basic ß-crystallins. Molecular dynamic simulations revealed that L116P mutation altered the hydrophobic residues at the surface around the mutant site, providing solvents more access to the internal and hydrophobic parts of the protein. CONCLUSIONS: Decreased ßB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with ßA3 protects against the low thermal stability of ßB1-L116P.

New insights into change of lens proteins' stability with ageing under physiological conditions.

BACKGROUND: Age-related cataract, which presents as a cloudy lens, is the primary cause of vision impairment worldwide and can cause more than 80% senile blindness. Previous studies mainly explored the profile of lens proteins at a low concentration because of technical limitations, which could not reflect physiological status. This study focuses on protein stability changes with ageing under physiological conditions using a novel equipment, Unchained Labs (Uncle), to evaluate protein thermal stability. METHODS: Samples were assessed through Unchained Labs, size-exclusion chromatography, western blot and biophysics approaches including the Thioflavin T, ultraviolet and internal fluorescence. RESULTS: With age, the melting temperature value shifted from 67.8°C in the young group to 64.2°C in the aged group. Meanwhile, crystallin may form more isomeric oligomers and easy to be degraded in aged lenses. The spectroscopic and size-exclusion chromatography results show a higher solubility after administrated with lanosterol under the environmental stress. CONCLUSION: We are the first to explore rabbit lens protein stability changes with ageing using biophysical methods under physiological conditions, and this study can conclude that the structural stability and solubility of lens proteins decrease with ageing. Additionally, lanosterol could aid in resolving protein aggregation, making it a potential therapeutic option for cataracts. So, this study provides cataract models for anti-cataract drug developments.

Heterozygous variants c.781G>A and c.1066dup of serine protease 56 cause familial nanophthalmos by impairing serine-type endopeptidase activity.

BACKGROUND/AIMS: Nanophthalmos is a rare developmental, bilateral, sporadic or hereditary form of microphthalmos. In this study, the heterozygous variants c.781G>A and c.1066dup of the PRSS56 gene were identified in two patients with nanophthalmos. This study reports the clinical manifestation and the underlying pathogenic mechanism. METHODS: Whole-exome sequencing was performed to identify the pathogenic genes in a Chinese family with nanophthalmos. The molecular simulation was used to predict the structures of wild-type or mutant PRSS56. The PRSS56 wild-type or mutation overexpression cellular models have been constructed accordingly. The subcellular localisation was then observed using immunofluorescence and Western-blot techniques. The Folin-Ciocalteu assay was carried out to evaluate serine-type endopeptidase activity, and a wound-healing assay was used to examine the cellular migratory ability. RESULTS: The whole-exome sequencing revealed that heterozygous variants c.781G>A and c.1066dup of the PRSS56 gene might contribute to nanophthalmos. Both variants were not identified in the dbSNP, 1000 Genome project or ESP6500 databases. Furthermore, the variants were highly conserved and were involved in biological functions. The mutations result in destructive protein structure and impede serine-type endopeptidase activity, thereby impairing subcellular localisation and cellular migration. CONCLUSION: The c.781G>A and c.1066dup variants of the PRSS56 gene might negatively affect protein structures, subcellular localisation, serine-type endopeptidase activity and cellular migratory ability. Together, these changes could lead to the development of nanophthalmos. This study identifies the PRSS56 gene as a potential target for nanophthalmos diagnosis and treatment.

Recent Advances of Intraocular Lens Materials and Surface Modification in Cataract Surgery.

Advances in cataract surgery have increased the demand for intraocular lens (IOL) materials. At present, the progress of IOL materials mainly contains further improving biocompatibility, providing better visual quality and adjustable ability, reducing surgical incision, as well as dealing with complications such as posterior capsular opacification (PCO) and ophthalmitis. The purpose of this review is to describe the research progress of relevant IOL materials classified according to different clinical purposes. The innovation of IOL materials is often based on the common IOL materials on the market, such as silicon and acrylate. Special properties and functions are obtained by adding extra polymers or surface modification. Most of these studies have not yet been commercialized, which requires a large number of clinical trials. But they provide valuable thoughts for the optimization of the IOL function.

A novel cataract-causing mutation Ile82Met of γA crystallin trends to aggregate with unfolding intermediate.

Cataract is the most common pathogenic ophthalmic disease leading to blindness in children worldwide. Genetic disorder is the leading cause of congenital cataract, among which crystallin mutations have a high incidence. There are few reports on γA-crystallin, one critical member of crystallin superfamilies. In this study, we identified a novel pathogenic mutation (Ile82Met) in γA-crystallin from a three-generation Chinese family with cataract, and investigated the potential molecular mechanism in detail. To elucidate the pathogenic mechanism of I82M mutant, spectroscopic and solubility experiments were performed to determine the difference between the purified γA-crystallin wild type (WT) and I82M mutant under both physiological conditions and environmental stresses (UV irradiation, thermal denaturation or chemical denaturation). The I82M mutant did not affect the secondary/tertiary structure of monomeric γA-crystallin under physiological status, but decreased protein stability and increased aggregatory potency under the stressful treatment. Surprisingly, the chemical denaturation caused I82M to switch from the two-state unfolding of γA-crystallin to three-state unfolding involving an unfolding intermediate. This study expands the genetic variation map of cataract, and provides novel insights into the pathomechanism, in particular, filling in a gap in the understanding of γA-crystallin mutants causing cataract.

Cataract-Causing S93R Mutant Destabilized Structural Conformation of ßB1 Crystallin Linking With Aggregates Formation and Cellular Viability.

Cataract, opacity of the eye lens, is the leading cause of visual impairment worldwide. The crucial pathogenic factors that cause cataract are misfolding and aggregation of crystallin protein. ßB1-crystallin, which is the most abundant water-soluble protein in mammalian lens, is essential for lens transparency. A previous study identified the missense mutation ßB1-S93R being responsible for congenital cataract. However, the exact pathogenic mechanism causing cataract remains unclear. The S93 residue, which is located at the first Greek-key motif of ßB1-crystallin, is highly conserved, and its substitution to Arginine severely impaired hydrogen bonds and structural conformation, which were evaluated via Molecular Dynamic Simulation. The ßB1-S93R was also found to be prone to aggregation in both human cell lines and Escherichia coli. Then, we isolated the ßB1-S93R variant from inclusion bodies by protein renaturation. The ßB1-S93R mutation exposed more hydrophobic residues, and the looser structural mutation was prone to aggregation. Furthermore, the S93R mutation reduced the structural stability of ßB1-crystallin when incubated at physiological temperature and made it more sensitive to environmental stress, such as UV irradiation or oxidative stress. We also constructed a ßB1-S93R cellular model and discovered that ßB1-S93R was more sensitive to environmental stress, causing not only aggregate formation but also cellular apoptosis and impaired cellular viability. All of the results indicated that lower solubility and structural stability, sensitivity to environmental stress, vulnerability to aggregation, and impaired cellular viability of ßB1-S93R might be involved in cataract development.

Safety of femtosecond laser-assisted cataract surgery versus conventional phacoemulsification for cataract: A meta-analysis and systematic review.

Purpose: To compare the complications of femtosecond laser-assisted cataract surgery (FLACS) with those of conventional phacoemulsification surgery (CPS) for age-related cataracts. Methods: PubMed, Cochrane Library, and EMBASE were systematically searched for studies comparing FLACS and CPS. Outcomes were operative complications, including the intraoperative capsule tear, postoperative corneal edema, macular edema, uncontrolled IOP, etc. The effect measures were weighted with odds ratios with 95% CIs. Results: Nineteen RCTs and 18 cohort studies, including 24,806 eyes (11,375 of the FLACS group and 13,431 of the CPS group), were identified. There were no significant differences between the two groups in anterior capsule tear, corneal edema, macular edema, uncontrolled IOP, vitreous loss, posterior vitreous detachment, etc. Posterior capsule tear rate showed a significantly lower in RCT subgroups (P â€‹= â€‹0.04) and without differences in total (P â€‹= â€‹0.63). Significant differences were observed in the incidence of descemet membrane tear/trauma (P â€‹= â€‹0.02) and IFIS/iris trauma (P â€‹= â€‹0.04. Additionally, The FLACS specific complications showed a significantly higher rate of miosis (P â€‹< â€‹0.0001), corneal epithelial defect (P â€‹= â€‹0.001), corneal haze (P â€‹= â€‹0.002), and subconjunctival hemorrhage (P â€‹= â€‹0.01). Conclusions: FLACS maintains the same safety compared with CPS in terms of all intraoperative and postoperative complications. Although FLACS did show a statistically significant difference for several FLACS specific complications, it would not influence the visual outcome and heal itself.

Cataract-causing mutation R48C increases γA-crystallin susceptibility to oxidative stress and ultraviolet radiation.

Among all congenital cataracts caused by genetic mutations, approximately half are caused by a mutation in crystallin genes, and accounts the leading cause of blindness in children globally. In this study, we investigated the underlying molecular mechanism of R48C mutation (c.142C > T; p.[Arg48Cys]) of γA-crystallin in a Mexican-Mestizo descent family causing congenital cataracts. We purified γA-crystallin wild-type (WT) and R48C mutant and compared their structural characteristics and biophysical properties by Spectroscopic experiments and environmental stress (oxidative stress, ultraviolet irradiation, pH disorders, thermal shock, or chemical denaturation). The R48C mutant did not affect the secondary and tertiary structure of monomer γA-crystallin, nor did it affect its stability to heat shock and chemicals. However, the R48C mutant destroys the oxidative stability of γA-crystallin, which makes the protein more prone to aggregation and precipitation under oxidative conditions. These might be the pathogenesis of γA-crystallin R48C mutant related to congenital cataract and help to develop anti-cataract strategies from the perspective of γA-crystallin.

Congenital cataract-causing mutation ßB1-L116P is prone to amyloid fibrils aggregation and protease degradation with low structural stability.

Congenital cataract, a common disease with lens opacification, causes blindness in the newborn worldwide and is mainly caused by abnormal aggregation of crystallin. As the main structural protein in the mammalian lens, ßB1-crystallin has an important role in the maintenance of lens transparency. Recently, the L116P mutation in ßB1-CRY was found in a Chinese family with congenital nuclear cataracts, while its underlying pathogenic mechanism remains unclear. In the current study, the ßB1 wild-type protein was purified, and the mutated form, ßB1-L116P, was examined for examining the effect on structural stability and susceptibility against environmental stresses. Our results reveal low solubility and structural stability of ßB1-L116P at physiological temperature, which markedly impaired the protein structure and the oligomerization of ßB1-crystallin. Under guanidine hydrochloride-induced denaturing conditions, ßB1-L116P mutation perturbed the protein unfolding process, making it prone to amyloid fibrils aggregation. More importantly, the L116P mutation increased susceptibility of ßB1-crystallin against UV radiation. ßB1-L116P overexpression led to the formation of more serious intracellular aggresomes under UV radiation or oxidative stress. Furthermore, the ßB1-L116P mutation increased the sensitivity to the proteolysis process. These results indicate that the low structural stability, susceptibility to amyloid fibrils aggregation, and protease degradation of ßB1-L116P may contribute to cataract development and associated symptoms.

Cataract-causing G91del mutant destabilised ßA3 heteromers formation linking with structural stability and cellular viability.

BACKGROUND/AIMS: Congenital cataracts, which are genetically heterogeneous eye disorders, result in visual loss in childhood around the world. CRYBA1/BA3 serves as an abundant structural protein in the lens, and forms homomers and heteromers to maintain lens transparency. In previous study, we identified a common cataract-causing mutation, ßA3-glycine at codon 91 (G91del) (c.271-273delGAG), which deleted a highly conserved G91del and led to perinuclear zonular cataract. In this study, we aimed to explore the underlying pathogenic mechanism of G91del mutation. METHODS: Protein purification, size-exclusion chromatography, spectroscopy and molecular dynamics simulation assays were used to investigate the effects on the heteromers formation and the protein structural properties of ßA3-crystallin caused by G91del mutation. Intracellular ßA3-G91del overexpression, MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) and cell apoptosis were used to investigate the cellular functions of ßA3-G91del. RESULTS: ßA3-crystallin and ßB2-crystallin could form heteromers, which have much more stable structures than ßA3 homomers. Interestingly, ßA3/ßB2 heteromers improved their resistance against the thermal stress and the guanidine hydrochloride treatment. However, the pathogenic mutation ßA3-G91del destroyed the interaction with ßB2, and thereby decreased its structural stability as well as the resistance of thermal or chemical stress. What's more, the ßA3-G91del mutation induced cell apoptosis and escaped from the protection of ßB2-crystallin. CONCLUSIONS: ßA3/ßB2 heteromers play an indispensable role in maintaining lens transparency, while the ßA3-G91del mutation destabilises heteromers formation with ßB2-crystallin, impairs cellular viability and induces cellular apoptosis. These all might contribute to cataract development.

ATP Can Efficiently Stabilize Protein through a Unique Mechanism.

Recent experiments suggested that ATP can effectively stabilize protein structure and inhibit protein aggregation when its concentration is less than 10 mM, which is significantly lower than cosolvent concentrations required in conventional mechanisms. The ultrahigh efficiency of ATP suggests a unique mechanism that is fundamentally different from previous models of cosolvents. In this work, we used molecular dynamics simulation and experiments to study the interactions of ATPs with three proteins: lysozyme, ubiquitin, and malate dehydrogenase. ATP tends to bind to the surface regions with high flexibility and high degree of hydration. These regions are also vulnerable to thermal perturbations. The bound ATPs further assemble into ATP clusters mediated by Mg2+ and Na+ ions. More interestingly, in Mg2+-free ATP solution, Na+ at higher concentration (150 mM under physiological conditions) can similarly mediate the formation of the ATP cluster on protein. The ATP cluster can effectively reduce the fluctuations of the vulnerable region and thus stabilize the protein against thermal perturbations. Both ATP binding and the considerable improvement of thermal stability of ATP-bound protein were verified by experiments.