Protein Thermal Stability Research Articles

Oxidoreductase disulfide bond proteins DsbA and DsbB form an active redox pair in Chlamydia trachomatis, a bacterium with disulfide dependent infection and development.

Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and—using recombinant purified proteins—demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein’s thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB differentiation coincided with the oxidation of the chlamydial outer membrane complex (COMC). Together with our demonstration of an active redox pairing, our findings suggest a potential role for CtDsbA and CtDsbB in the critical disulfide bond formation step in the highly regulated development cycle.

Size-dependent macromolecular crowding effect on the thermodynamics of protein unfolding revealed at the single molecular level

The real biological environment involves a high degree of complexity and the macromolecular crowder is the best candidate to somewhat mimic this. In this contribution, we have used two different sized dextrans as model crowders and human serum albumin (HSA) as a model protein to decipher how the thermal stability of protein is modulated inside the crowded milieu and also to understand the effect of the size of the crowders. In our previous report (Biochemistry 2018, 57, 6078–6089) we have proposed the presence of some interaction between dextran-6 and HSA, which are probably not present between the larger dextrans and HSA. Complete thermodynamic analysis of thermal denaturation profile of HSA suggests that small crowders increase protein stability mainly via the enthalpy of denaturation while larger crowders increase stability primarily through entropy. Further, the active site dynamics is altered significantly in the presence of larger dextran-40, but not by smaller dextran-6. Surprisingly, the dynamics of the more compact intermediate state does not get modified by the crowders. Overall, our result indicates that biomacromolecules of similar chemical composition and shape may exert their effect not only by different extent but also by a different mechanism, owing to their different sizes.

QresFEP: An Automated Protocol for Free Energy Calculations of Protein Mutations in Q.

Predicting the effect of single-point mutations on protein stability or protein-ligand binding is a major challenge in computational biology. Free energy calculations constitute the most rigorous approach to this problem, though the estimation of converged values for amino acid mutations remains challenging. To overcome this limitation, we developed tailored protocols to calculate free energy shifts associated with single-point mutations. We herein describe the QresFEP protocol, which includes an extension of our recent protocols to cover all amino acids mutations, based on the latest versions of the OPLS-AA force field. QresFEP is implemented in an application programming interface framework and the graphic interface QGui, for the molecular dynamics software Q. The complete protocol is benchmarked in several model systems, optimizing a number of sampling parameters and the implementation of Zwanzig's exponential formula and Bennet's acceptance ratio methods. QresFEP shows an excellent performance on estimating the hydration free energies of amino acid side-chain mimics, including their charged analogues. We also examined its performance on a protein-ligand binding problem of pharmaceutical relevance, the antagonism of neuropeptide Y1 G protein-coupled receptor. Here, the calculations show very good agreement with the experimental effect of 16 mutations on the binding of antagonists BIBP3226, in line with our recent applications in this field. Finally, the characterization of 43 mutations of T4-lysozyme reveals the capacity of our protocol to assess variations of the thermal stability of proteins, achieving a similar performance to alternative free energy perturbation (FEP) approaches. In summary, QresFEP is a robust, versatile, and user-friendly computational FEP protocol to examine biochemical effects of single-point mutations with high accuracy.

SimpleDSFviewer: A tool to analyze and view differential scanning fluorimetry data for characterizing protein thermal stability and interactions.

Differential scanning fluorimetry (DSF) is a widely used thermal shift assay for measuring protein stability and protein-ligand interactions that are simple, cheap, and amenable to high throughput. However, data analysis remains a challenge, requiring improved methods. Here, the program SimpleDSFviewer, a user-friendly interface, is described to help the researchers who apply DSF technique in their studies. SimpleDSFviewer integrates melting curve (MC) normalization, smoothing, and melting temperature (Tm) analysis and directly previews analyzed data, providing an efficient analysis tool for DSF. SimpleDSFviewer is developed in Matlab, and it is freely available for all users to use in Matlab workspace or with Matlab Runtime. It is easy to use and an efficient tool for researchers to preview and analyze their data in a very short time.

Structural and mutational investigation of psychrophilic adenylate kinase reveals the importance of hydrophobic packing in protein thermal stability

Structural and mutational investigation of psychrophilic adenylate kinase reveals the importance of hydrophobic packing in protein thermal stability

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

Solution thermodynamic approach to analyze protein stability in aqueous solutions

Protein thermal stability was analyzed by a solution thermodynamic approach. The small energetic differences in hydrogen-bonds (HB) among amino acid resdues and water molecules were proved to be amplified by the large number of HB involved to bring about the equilibrium shift from folding to unfolding of proteins. In aqueous solutions, water activity (Aw) plays a key role in protein stability. Therefore, Aw was precisely determined for various solutions and its relationship with solution structure was discussed. Wyman-Tanford analysis based on Aw showed linear regressions, without exception, between protein unfolding-ratio and Aw for lysozyme, ribonuclease A, and α-chymotrypsinogen A in various solutions with sugars, osmolytes, alcohols, and protein denaturant. From this linear regression, the free energy difference, ΔΔG, for a protein in a solution and in pure water, was easily obtained. Protein stability in a solution was proved to be determined by a balance between hydration and solute-binding effects to the protein and also by solution structure, which indirectly affects the hydrophobic interaction in a protein molecule. Temperature dependence of HB on protein stability suggested its interrelationship with hydrophobic interaction.

Probing Thermal Stability of Proteins with Temperature Scanning Viscometer.

Thermal stability is essential for the understanding of protein stability and is a critical quality attribute of therapeutic biologics, including enzymes, fusion proteins, monoclonal antibodies, etc. The commonly used analytical methods, such as differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), and circular dichroism (CD), have their limitations in measuring protein thermal stability. Through this work, we described a novel method to probe the thermal stability of proteins in various formulations using a temperature scanning viscometer. The viscosity of the material was plotted against the temperature, and the peak in the first derivative of the viscosity versus temperature was shown to be related to the protein melting temperature. The measured melting temperature of bovine serum albumin (BSA) at a concentration of 1 mg/mL in phosphate buffer was 63 °C, which was close to the value of 64 °C obtained by DSC. The unfolding of BSA was confirmed using orthogonal techniques of second derivative ultraviolet-visible (UV-vis) spectroscopy and dynamic light scattering (DLS). This method was also able to reveal the microenvironment changes of proteins, including formulation effects. Other multiple domains proteins including lysozyme and IgG were also tested using this method and showed comparable melting temperatures with DSC. This work showed the feasibility of using a temperature scanning viscometer to measure the thermal stability of proteins in diverse formulation matrices with wider protein concentration ranges.

Hydrophobic surface residues can stabilize a protein through improved water-protein interactions.

Protein stabilization is difficult to rationalize, but the detailed thermodynamic and structural analysis of a series of carefully designed mutants mayprovide experimental insights into the mechanisms underlying stabilization. Here, we report a systematic structural and thermodynamic analysis of bovine pancreatic trypsin inhibitor (BPTI) variants that are significantly stabilized through a single amino acid substitution at residue 38, which is located in a loop mostly exposed on the protein surface. Differential scanning calorimetry indicated that the BPTI-[5,55]Gly14 variantswith a single mutation at position 38 were stabilized in an enthalpy-driven manner and that the magnitude of the stabilization increased as the hydrophobicity of residue 38 increased. This increase in the thermal stability of BPTI was unexpected because a hydrophobic residue on a protein surface is usually destabilizing. To identify the structural determinants of this stabilization, we determined the crystalstructures of six BPTI-[5,55]Gly14 variants (Gly14 Gly38 , Gly14 Ala38 , Gly14 Val38 , Gly14 Leu38 , Gly14 Ile38 , and Gly14 Lys38 )at high resolutions and showed that theyretain essentially the same structure as the wild-type BPTI. Amore detailed examination of their structuresindicated that the extent of thermal stabilization correlated with both improved local packing and increased hydration around the substitution sites. In particular, the number of water molecules near residue 38 increased upon mutation to a hydrophobic residue suggesting that improved hydration contributed to the enthalpy-driven stabilization. Increasing a protein's thermal stability by the placement of a hydrophobic amino acid on the protein surface is a novel and unexpected phenomenon, and its exact nature is worth further examination, as it may provide a generic method for stabilizing proteins in an enthalpy-driven manner. DATABASE: The coordinates and structure factors of Gly14 Gly38 , Gly14 Ile38 , Gly14 Leu38 , and Gly14 Lys38 variants of BPTI-[5,55] are deposited in the Protein Data Bank under the PDB entry codes 5XX3, 5XX5, 5XX2, and 5XX4, respectively. We previously reported the structures of Gly14 Ala38 (2ZJX) and Gly14 Val38 (2ZVX).

Novel non-linear curve fitting to resolve protein unfolding transitions in intrinsic fluorescence differential scanning fluorimetry

In biotherapeutic protein research, an estimation of the studied protein’s thermal stability is one of the important steps that determine developability as a function of solvent conditions. Differential Scanning Fluorimetry (DSF) can be applied to measure thermal stability. Label-free DSF measures amino acid fluorescence as a function of temperature, where conformational changes induce observable peak deformation, yielding apparent melting temperatures. The estimation of the stability parameters can be hindered in the case of multidomain, multimeric or aggregating proteins when multiple transitions partially coincide. These overlapping protein unfolding transitions are hard to evaluate by the conventional methodology, as peak maxima are shifted by convolution. We show how non-linear curve fitting of intrinsic fluorescence DSF can deconvolute highly overlapping transitions in formulation screening in a semi-automated process. The proposed methodology relies on synchronous, constrained fits of the fluorescence intensity, ratio and their derivatives, by combining linear baselines with generalized logistic transition functions. The proposed algorithm is applied to data from three proteins; a single transition, a double separated transition and a double overlapping transition. Extracted thermal stability parameters; apparent melting temperatures Tm,1, Tm,2 and melting onset temperature Tonset are obtained and compared with reference software analysis. The fits show R2 = 0.94 for single and R2 = 0.88 for separated transitions. Obtaining values and trends for Tonset in a well-described and automated way, will aid protein scientist to better evaluate the thermal stability of proteins.

APPLICATION OF A TWO-STAGE AGITATION SPEED CONTROL STRATEGY TO ENHANCE YELLOW PIGMENTS PRODUCTION BY MONASCUS ANKA MUTANT

Monascus anka mutant strain was isolated using conventional mutation techniques in our laboratory. In our previous study, strain Monascus anka mutant showed high yield of yellow pigments with the characteristics of high protein adhesion, thermal stability, and wide-pH stability. This work focused on the agitation optimization in a 5 L fermentor of strain Monascus anka mutant and two stage agitation speed control strategy was operated as follows: the agitation speed was 400 r/min to improve the Monascus growth and yellow pigments production before culturing 96 hour and then 300 r/min was carried out to extend the time of yellow pigment production with efficient using substrate. The yield of monascus yellow pigments reached 149.43 OD, which was 49.37%，49.25%，35.56%，18.73%, and 41.01% higher than that of 250 r/min, 300 r/min, 350 r/min, 400 r/min, and 450 r/min fermentation, respectively. The maximum specific growth rate (µmax) and the maximum specific production rate of yellow pigments (qy) reached 0.0528 h-1 and 0.2177 OD.g-1.h-1, respectively. Specific growth rate (µ) maintained higher than 0.01 h-1 from 6 hours to 54 hours, and qy could maintain at 0.10 OD.g-1.h-1 from 24 hour to 66 hour, which could not be obtained by single agitation control. The data of this work could contribute to making the industrial production of Monascus yellow pigments feasible.

A Core-Shell Structured Immobilized Lipase and Its Application in High-Temperature Reactions.

In this research, a core-shell immobilized lipase was constructed with the cost-effective bamboo carrier. Natural bamboo powder was pretreated by different methods. The results of SEM and BET demonstrated that alkali pretreatment increased the surface roughness with formed wrinkles and micropores, facilitating the lipase adsorption on the pretreated carrier. The optimal immobilization conditions were obtained through the response surface method, and the optimized conditions were 2wt% sodium hydroxide solution, 1.2wt% sodium alginate, and 0.69wt% calcium chloride at a calcification time of 100min. Under this condition, the immobilized lipase showed an excellent protein adsorption rate and thermal stability. When the core-shell structured immobilized lipase was used in wax ester synthesis system, the esterification yield (80%) did not show an obvious decline after 17 batches. The results indicated that the core-shell immobilized lipase is effective for the biocatalytic reaction, and the immobilization method is a promising strategy for future enzyme immobilization and modification in industrial applications.

Thermal stability and secondary structure of feruloylated Rapana thomasiana hemocyanin

This is the first report on the conjugation of a hemocyanin from Rapana thomasiana (RtH) with ferulic acid, which itself exhibits an immunostimulatory activity. In a two-step reaction, 20 to 180 ferulic acid residues were covalently linked to one molecule RtH. The effect of modification of RtH with ferulic acid on the thermal unfolding was evaluated using differential scanning calorimetry (DSC). The DSC curves of the feruloylated-RtHs characterize with an asymmetric shape, which is an indication for the existence of more than one structural unit in the analyzed samples. The protein thermal stability is not affected by the modification; however, the profiles of the DSC curves of the feruloylated-RtH and the native RtH differ, which implies the reorganization in the protein molecule and is in correlation with the secondary structure analyses. The conformational changes in the RtH molecules that are due to the feruloylation were followed in the Amide I region (1600–1700 cm−1) of the ATR-FTIR spectra. In all prepared samples, we observed a rearrangement in protein molecule, decrease in α-helices and coiled structures in favor of the β-structures, and no aggregation in comparison with the native RtH.

Effect of protein thermal stability and protein secondary structure on the roasted mutton texture and colour from different cuts

The age and cut of mutton are important factors influencing the suitability of roasting. In this study, the sensory quality, texture profile and surface colour of roasted mutton cuts (back strap, silverside, square cut shoulder, breast and flap and oyster cut) from Merino sheep slaughtered at ages of six months and two years were investigated. Also the protein thermal stability and protein secondary structure of different samples were also evaluated. The results showed that the muscles of back strap, silverside and oyster cut were much more suitable for roasting, presented higher sensory scores and better corresponding instrumental colour, texture properties and shear force. Lower protein thermal stability of collagen, sarcoplasmic proteins and actin in raw muscle might be the cause of the higher values of sensory attributes, L⁎, texture profiles and lower a⁎ value of roasted mutton. The lower relative percentage of protein α-helix in fresh mutton might contribute to the higher protein random coil of roasted mutton, and these contribute with a great texture and colour. It concludes that differential protein thermal stability and protein secondary structure of muscles partially affect the roast quality of lamb.

Mimicking Molecular Chaperones to Regulate Protein Folding.

Folding and unfolding are essential ways for a protein to regulate its biological activity. The misfolding of proteins usually reduces or completely compromises their biological functions, which eventually causes a wide range of diseases including neurodegeneration diseases, type II diabetes, and cancers. Therefore, materials that can regulate protein folding and maintain proteostasis are of significant biological and medical importance. In living organisms, molecular chaperones are a family of proteins that maintain proteostasis by interacting with, stabilizing, and repairing various non-native proteins. In the past few decades, efforts have been made to create artificial systems to mimic the structure and biological functions of nature chaperonins. Herein, recent progress in the design and construction of materials that mimic different kinds of natural molecular chaperones is summarized. The fabrication methods, construction rules, and working mechanisms of these artificial chaperone systems are described. The application of these materials in enhancing the thermal stability of proteins, assisting de novo folding of proteins, and preventing formation of toxic protein aggregates is also highlighted and explored. Finally, the challenges and potential in the field of chaperone-mimetic materials are discussed.

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca2+-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.

Determining the Protein Stability of Alzheimer's Disease Protein, Amyloid Precursor Protein.

Determining protein thermal stability is integral in biomedical research. Here, with the use of two thermal stability assays, we show the melting temperature of amyloid precursor protein, an Alzheimer's disease related protein. The average melting temperature for amyloid precursor protein of 55.9°C was derived from differential scanning fluorometry (55.1 ± 0.3°C) and cellular thermal melt (56.7 ± 0.7°C). These experimental methods have significant application for Alzheimer's disease research including their use for amyloid precursor protein stability profiling and for the identification of additional binding partners to further elucidate novel protein functions.

Assessment of HSP70 and catalase in Brachidontes rodriguezii (d’Orbigny, 1842) a mussel from the Argentinean coast

Introduction: Intertidal ecosystems are complex environments of great importance for the ecological balance of coastal zones and are vulnerable areas to effects caused by natural stressors of global relevance such as temperature. Mussels, are often exposed to drastic variations of temperature and can show intracellular mechanisms of compensation that allow them to counteract exposure in the short and medium term at high temperatures. Heat shock proteins are chaperones that allow thermal stability of proteins against abrupt changes in temperature and catalase is an antioxidant enzyme that aid cells to fight against oxidative stress caused by diverse environmental stressors [1,2]. Materials and methods: Mussel samples were homogenized in PBS buffer (phosphate saline solution, pH =7.4) and centrifuged (10,000×g for 15 min at 4 °C). Afterwards, the supernatants were transferred to 1.5 ml microtubes and stored at −80 °C until analysis. The HSP70 (Heat shock Proteins) levels were determined in individuals from both sexes, exposed at different temperatures (20 °C, 24 °C, 26 °C y 28 °C) for 7 and 14 days. Organisms sampled at T0 were also analysed. The HSP70 levels were determined by ELISA (Enzyme Linked Immunosorbent Assay) as described by Madeira et al. [2] and catalase following the method described in Diniz et al. [3]. Results and discussion: The preliminary results show an absence of significant differences between sexes of HSP70 levels at T0, 20 °C and 24 °C. However, at 26 °C and 28 °C, a differentiated response was observed in organisms exposed at higher temperatures, where the females had higher levels of HSP70 in the short term (7 days) compared to male individuals (p <0.05). In the medium term (14 days) it was observed that females have similar or lower values than males. Conclusions: The results show the ability to evaluate thermal stress by determining biomarkers such as HSP70 or antioxidant enzymes in tissues of B. rodriguezii providing valuable information to a better understanding of the effects of natural stressors on marine organisms. It also establishes that the sex of animals is a major factor of importance when evaluating this type of responses related to thermal stress.

Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

Adenosine triphosphate (ATP) plays fundamental roles in cellular biochemistry and was recently discovered to function as a biological hydrotrope. Here, we use mass spectrometry to interrogate ATP-mediated regulation of protein thermal stability and protein solubility on a proteome-wide scale. Thermal proteome profiling reveals high affinity interactions of ATP as a substrate and as an allosteric modulator that has widespread influence on protein complexes and their stability. Further, we develop a strategy for proteome-wide solubility profiling, and discover ATP-dependent solubilization of at least 25% of the insoluble proteome. ATP increases the solubility of positively charged, intrinsically disordered proteins, and their susceptibility for solubilization varies depending on their localization to different membrane-less organelles. Moreover, a few proteins, exhibit an ATP-dependent decrease in solubility, likely reflecting polymer formation. Our data provides a proteome-wide, quantitative insight into how ATP influences protein structure and solubility across the spectrum of physiologically relevant concentrations.

Structural and mutational analyses of psychrophilic and mesophilic adenylate kinases highlight the role of hydrophobic interactions in protein thermal stability.

Protein thermal stability is an important field since thermally stable proteins are desirable in many academic and industrial settings. Information on protein thermal stabilization can be obtained by comparing homologous proteins from organisms living at distinct temperatures. Here, we report structural and mutational analyses of adenylate kinases (AKs) from psychrophilic Bacillus globisporus (AKp) and mesophilic Bacillus subtilis (AKm). Sequence and structural comparison showed suboptimal hydrophobic packing around Thr26 in the CORE domain of AKp, which was replaced with an Ile residue in AKm. Mutations that improved hydrophobicity of the Thr residue increased the thermal stability of the psychrophilic AKp, and the largest stabilization was observed for a Thr-to-Ile substitution. Furthermore, a reverse Ile-to-Thr mutation in the mesophilic AKm significantly decreased thermal stability. We determined the crystal structures of mutant AKs to confirm the impact of the residue substitutions on the overall stability. Taken together, our results provide a structural basis for the stability difference between psychrophilic and mesophilic AK homologues and highlight the role of hydrophobic interactions in protein thermal stability.