Molecular Modeling of the Multiple-Substrate Activity of the Human Recombinant Intra-Melanosomal Domain of Tyrosinase and Its OCA1B-Related Mutant Variant P406L.

Tyrosinase serves as the key enzyme in melanin biosynthesis, catalyzing the initial steps of the pathway, the hydroxylation of the amino acid L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), followed by the subsequent oxidation of L-DOPA into dopaquinone (DQ), and it facilitates the conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into 5,6-indolequinone-2-carboxylic acid (IQCA) and 5,6-dihydroxy indole (DHI) into indolequinone (IQ). Despite its versatile substrate capabilities, the precise mechanism underlying tyrosinase's multi-substrate activity remains unclear. Previously, we expressed, purified, and characterized the recombinant intra-melanosomal domain of human tyrosinase (rTyr). Here, we demonstrate that rTyr mimics native human tyrosinase's catalytic activities in vitro and in silico. Molecular docking and molecular dynamics (MD) simulations, based on rTyr's homology model, reveal variable durability and binding preferences among tyrosinase substrates and products. Analysis of root mean square deviation (RMSD) highlights the significance of conserved residues (E203, K334, F347, and V377), which exhibit flexibility during the ligands' binding. Additionally, in silico analysis demonstrated that the OCA1B-related P406L mutation in tyrosinase substantially influences substrate binding, as evidenced by the decreased number of stable ligand conformations. This correlation underscores the mutation's impact on substrate docking, which aligns with the observed reduction in rTyr activity. Our study highlights how rTyr dynamically adjusts its structure to accommodate diverse substrates and suggests a way to modulate rTyr ligand plasticity.

In vitro characterization of the intramelanosomal domain of human recombinant TYRP1 and its oculocutaneous albinism type 3-related mutant variants.

Tyrosinase related protein 1 (TYRP1) is the most abundant melanosomal protein of the melanocyte, where plays an important role in the synthesis of eumelanin, possibly catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid to 5,6-quinone-2-carboxylic acid. Mutations to the TYRP1 gene can result in oculocutaneous albinism type 3 (OCA3), a rare disease characterized by reduced synthesis of melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the intramelanosomal domain of TYRP1 and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in tyrosinase. Proteins were produced in Trichoplusia Ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich domain show the variants misfolding during the purification process. The H215Y variant disrupts the binding of Zn2+ in the active site and thus reduces the strength of the enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the protein surface are expected to have a negligible phenotype change compared to that of TYRP1. For the mutations with severe phenotype changes, which were localized in the Cys-rich domain or the active site, we confirmed a complete or partial protein misfolding as the possible mechanism of protein malfunction caused by OCA3 inherited mutations.

Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants.

Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene. Here, for the first time, we characterized the intra-melanosomal protein domain of Tyrp2 (residues 1-474) and missense variants C40S and C61W, which mimic the alterations found in genetic studies. Recombinant proteins were produced in the Trichoplusia Ni (Ti. Ni) larvae, purified by a combination of immobilized metal affinity (IMAC) and gel-filtration (GF) chromatography, and biochemically characterized. The mutants showed the protein expression in the lysates such as the wild type; however, undetectable protein yield after two steps of purification exhibited their misfolding and instability. In addition, the misfolding effect of the mutations was confirmed computationally using homology modeling and molecular docking. Together, experiments in vitro and computer simulations indicated the critical role of the Cys-rich domain in the Tyrp2 protein stability. The results are consistent with molecular modeling, global computational mutagenesis, and clinical data, proving the significance of genetic alterations in cysteine residues, which could cause oculocutaneous albinism type 8.

In Vitro Reconstitution of the Melanin Pathway's Catalytic Activities Using Tyrosinase Nanoparticles.

The melanogenesis pathway is characterized by a series of reactions catalyzed by key enzymes, such as tyrosinase (TYR), tyrosinase-related protein 2 (TYRP2), and tyrosinase-related protein 1 (TYRP1), to produce melanin pigment. However, in vitro studies of the catalytic activity were incomplete because of a lack of commercially available enzyme substrates, such as dopachrome. Herein, human recombinant intra-melanosomal domains of key enzymes were produced in Trichoplusia ni (T. ni) larvae and then purified using a combination of chromatography techniques in catalytically active form. Using Michaelis-Menten kinetics, the diphenol oxidase activity of tyrosinase achieved the maximum production of native dopachrome at 10 min of incubation at 37 °C for TYR immobilized to magnetic beads (TYR-MB). The presence of dopachrome was confirmed spectrophotometrically at 475 nm through HPLC analysis and in the TYRP2-catalyzed reaction, yielding 5,6-dihydroxyindole-2-carboxylic acid (DHICA). In the TYRP1-driven oxidation of DHICA, the formation of 5,6-indolequinone-2-carboxylic acid (IQCA) was confirmed at ~560 nm. This is the first in vitro reconstitution of the reactions from the melanogenic pathway based on intra-melanosomal domains. In the future, this approach could be used for quantitative in vitro analysis of the melanin pathway, biochemical effects associated with inherited disease-related mutations, and drug screens.

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding.

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.

Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase.

Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type 1 (OCA1). The mechanisms by which individual mutations contribute to the diverse pigmentation phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis-Menten and Van't Hoff analyses. Recombinant truncated human Tyr proteins (residues 19-469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction.

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity.

Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Here, we analyze protein binding using the diphenol oxidase behavior of Tyr and van 't Hoff temperature-dependent analysis. Recombinant Tyr was expressed and purified using a combination of affinity and size-exclusion chromatography. Michaelis-Menten constants were measured spectrophotometrically from diphenol oxidase reactions of Tyr, using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, at temperatures: 25, 31, 37, and 43 °C. Under the same conditions, the Tyr structure and the L-DOPA binding activity were simulated using 3 ns molecular dynamics and docking. The thermal Michaelis-Menten kinetics data were subjected to the van 't Hoff analysis and fitted with the computational model. The temperature-dependent analysis suggests that the association of L-DOPA with Tyr is a spontaneous enthalpy-driven reaction, which becomes unfavorable at the final step of dopachrome formation.

Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1.

Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.

The TYRP1-mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains.

Tyrosinases are melanocyte-specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra-melanosomal domains of tyrosinase, TYRtr (19-469), and tyrosinase-related protein 1, TYRP1tr (25-472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero-oligomeric complexes to maintain the protective function.

Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein.

Human tyrosinase (hTyr) is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Mutations in the Tyr gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Currently, the application of enzyme replacement therapy for a treatment of OCA1 is hampered by the absence of pure hTyr. Here, full-length hTyr (residues 1-529) was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the hTyr in detergent with the soluble recombinant intra-melanosomal domain, hTyrCtr (residues 19-469). Active hTyr is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, hTyr and hTyrCtr, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human tyrosinase potentially could be a useful tool in developing a cure for OCA1.

Dynamic analysis of human tyrosinase intra-melanosomal domain and mutant variants to further understand oculocutaneous albinism type 1.

Human tyrosinase (Tyr) is a Type I membrane glycoprotein that is the rate-limiting enzyme for controlling the production of melanin pigment in melanosomes. Currently, ~300 Tyr mutations are known to be involved in the genetic disease oculocutaneous albinism type 1 (OCA1), which exists in two forms, OCA1A and OCA1B. OCA1A is caused by a full loss of Tyr enzymatic activity, resulting in the absence of pigment in the skin, hair, and eyes, while OCA1B has reduced Tyr activity and pigment. Here, we used molecular modeling to try to understand the role of genetic changes at the protein level in inherited disease. The significant part of Tyr intra-melanosomal domain and five OCA1 mutant variants were built by homology modeling, glycosylated in silico, and refined using molecular dynamics in water. The modeling confirmed experimental results that N347 and N371 glycosylation is vital for protein stability. The changes caused by the T373K mutation indicate a significant impact on protein structure, as expected for OCA1A. In addition, evaluation of free energy changes in OCA1B mutants showed a strong association with the changes observed in our unfolding/refolding experiments in vitro. In conclusion, our results could be useful for understanding the role of OCA1 mutant variants in melanin pigment production, in silico searching for inhibitors and activators of tyrosinase activity, and genotype-to- phenotype analysis in OCA1.

Apoferritin is maintaining the native conformation of citrate synthase in vitro.

Ferritin, a member of a family of iron storage proteins, is expressed in conditions of oxidative or thermal stress in the cell. Ferritin widely found in human tissues including the eye and brain. Increased expression under oxidative or temperature stress conditions and protective effect on cell viability suggest that apo form of ferritin (apoferritin) may have a role in the formation or maintenance of the native conformation of proteins. To test this hypothesis, we studied the influence of apoferritin on the unfolding and refolding of citrate synthase (CS) in vitro. Here we show that at stoichiometric amounts apoferritin is remarkably protecting the CS catalytic activity, stabilize the aggregation of CS under heat stress and act as chaperone-like molecules in these folding reactions in vitro. Furthermore, apoferritin promote the functional refolding of CS after guanidinium hydrochloride denaturation. Finally, these results confirm that apoferritin has chaperone-like activity in vitro and suggests that apoferritin might have a role in protection and maintaining of protein native conformation.

The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase.

Tyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studied in vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residues in vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector.

The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc.

Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism.

Thermodynamic analysis of weak protein interactions using sedimentation equilibrium.

Proteins self-associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer-dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters, such as Gibbs free energy, enthalpy, and entropy, which can predict which major forces are involved in protein association.

Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rß2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rß2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

BACKGROUND: Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. METHODOLOGY/PRINCIPAL FINDINGS: The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. CONCLUSIONS/SIGNIFICANCE: The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin.

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.