RESUMEN

Disulfide bonds, covalently formed by sulfur atoms in cysteine residues, play a crucial role in protein folding and structure stability. Considering their significance, artificial disulfide bonds are often introduced to enhance protein thermostability. Although an increasing number of tools can assist with this task, significant amounts of time and resources are often wasted owing to inadequate consideration. To enhance the accuracy and efficiency of designing disulfide bonds for protein thermostability improvement, we initially collected disulfide bond and protein thermostability data from extensive literature sources. Thereafter, we extracted various sequence- and structure-based features and constructed machine-learning models to predict whether disulfide bonds can improve protein thermostability. Among all models, the neighborhood context model based on the Adaboost-DT algorithm performed the best, yielding "area under the receiver operating characteristic curve" and accuracy scores of 0.773 and 0.714, respectively. Furthermore, we also found AlphaFold2 to exhibit high superiority in predicting disulfide bonds, and to some extent, the coevolutionary relationship between residue pairs potentially guided artificial disulfide bond design. Moreover, several mutants of imine reductase 89 (IR89) with artificially designed thermostable disulfide bonds were experimentally proven to be considerably efficient for substrate catalysis. The SS-bond data have been integrated into an online server, namely, ThermoLink, available at guolab.mpu.edu.mo/thermoLink.

Asunto(s)

RESUMEN

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.

Asunto(s)

RESUMEN

Conventional statistical investigations have primarily focused on the comparison of the simple one-dimensional characteristics of protein cavities, such as number, surface area, and volume. These studies have failed to discern the crucial distinctions in cavity properties between thermophilic and mesophilic proteins that contribute to protein thermostability. In this study, the significance of cavity properties, i.e., flexibility and location, in protein thermostability was investigated by comparing structural differences between homologous thermophilic and mesophilic proteins. Three dimensions of protein structure were categorized into three regions (core, boundary, and surface) and a comparative analysis of cavity properties using this structural index was conducted. The statistical analysis revealed that cavity flexibility is closely related to protein thermostability. The core cavities of thermophilic proteins were less flexible than those of mesophilic proteins (averaged B' factor values, -0.6484 and -0.5111), which might be less deleterious to protein thermostability. Thermophilic proteins exhibited fewer cavities in the boundary and surface regions. Notably, cavities in mesophilic proteins, across all regions, exhibited greater flexibility than those in thermophilic proteins (>95% probability). The increased flexibility of cavities in the boundary and surface regions of mesophilic proteins, as opposed to thermophilic proteins, may compromise stability. Recent protein engineering investigations involving mesophilic xylanase and protease showed results consistent with the findings of this study, suggesting that the manipulation of flexible cavities in the surface region can enhance thermostability. Consequently, our findings suggest that a rational or computational approach to the design of flexible cavities in surface or boundary regions could serve as an effective strategy to enhance the thermostability of mesophilic proteins.

RESUMEN

BACKGROUND: Babesia orientalis is an intra-erythrocytic protozoan parasite that causes babesiosis in water buffalo. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three HSPs was developed in vitro. METHODS: BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay (cell proliferation assay based on thiazolyl blue tetrazolium bromide). RESULTS: Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 °C, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 °C heat shock. CONCLUSION: Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In conclusion, the in vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host-parasite interaction.

Asunto(s)

RESUMEN

Interactions between distantly placed amino acids in the primary chain (long-range) play a very crucial role in the formation and stabilization of the tertiary structure of a protein, while interactions between closely placed amino acids in the primary chain (short-range) mostly stabilize the secondary structures. Every protein needs to maintain marginal stability in order to perform its physiological functions in its native environment. The requirements for this stability in mesophilic and thermophilic proteins are different. Thermophilic proteins need to form more interactions as well as more stable interactions to survive in the extreme environment, they live in. Here, we aim to find out how the interacting amino acids in three-dimensional space are positioned in the primary chains in thermophilic and mesophilic. How does this arrangement help thermophiles to maintain their structural integrity at high temperatures? Working on a dataset of 1560 orthologous pairs we perceive that thermophiles are not only enriched with long-range interactions, they feature bigger connected clusters and higher network densities compared to their mesophilic orthologs, at higher interaction strengths between the amino acids. Moreover, we have observed the enrichment of different types of interactions at different secondary structural regions.

Asunto(s)

RESUMEN

CRISPR has revolutionized the field of genome editing in life sciences by serving as a versatile and state-of-the-art tool. Cas12f1 is a small nuclease of the bacterial immunity CRISPR system with an ideal size for cellular delivery, in contrast to CRISPR-associated (Cas) proteins like Cas9 or Cas12. However, Cas12f1 works best at low salt concentrations. In this study, we find that the plasticity of certain Cas12f1 regions (K196-Y202 and I452-L515) is negatively affected by increased salt concentrations. On this line, key protein domains (REC1, WED, Nuc, lid) that are involved in the DNA-target recognition and the activation of the catalytic RuvC domain are in turn also affected. We suggest that salt concentration should be taken in to consideration for activity assessments of Cas engineered variants, especially if the mutations are on the protospacer adjacent motif interacting domain. The results can be exploited for the engineering of Cas variants and the assessment of their activity at varying salt concentrations. We propose that the K198Q mutation can restore at great degree the compromised plasticity and could potentially lead to salt-tolerant Cas12f1 variants. The methodology can be also employed for the study of biomolecules in terms of their salinity tolerance.Communicated by Ramaswamy H. Sarma.

RESUMEN

Highly abundant proteins tend to evolve slowly (a trend called E-R anticorrelation), and a number of hypotheses have been proposed to explain this phenomenon. The misfolding avoidance hypothesis attributes the E-R anticorrelation to the abundance-dependent toxic effects of protein misfolding. To avoid these toxic effects, protein sequences (particularly those of highly expressed proteins) would be under selection to fold properly. One prediction of the misfolding avoidance hypothesis is that highly abundant proteins should exhibit high thermostability (i.e., a highly negative free energy of folding, ΔG). Thus far, only a handful of analyses have tested for a relationship between protein abundance and thermostability, producing contradictory results. These analyses have been limited by 1) the scarcity of ΔG data, 2) the fact that these data have been obtained by different laboratories and under different experimental conditions, 3) the problems associated with using proteins' melting energy (Tm) as a proxy for ΔG, and 4) the difficulty of controlling for potentially confounding variables. Here, we use computational methods to compare the free energy of folding of pairs of human-mouse orthologous proteins with different expression levels. Even though the effect size is limited, the most highly expressed ortholog is often the one with a more negative ΔG of folding, indicating that highly expressed proteins are often more thermostable.

Asunto(s)

RESUMEN

Thermal shift assay (TSA), also commonly designed by differential scanning fluorimetry (DSF) or ThermoFluor, is a technique relatively easy to implement and perform, useful in a myriad of applications. In addition to versatility, it is also rather inexpensive, making it suitable for high-throughput approaches. TSA uses a fluorescent dye to monitor the thermal denaturation of the protein under study and determine its melting temperature (Tm). One of its main applications is to identify the best buffers and additives that enhance protein stability.Understanding the TSA operating mode and the main methodological steps is a central key to designing effective experiments and retrieving meaningful conclusions. This chapter intends to present a straightforward TSA protocol, with different troubleshooting tips, to screen effective protein stabilizers such as buffers and additives, as well as data treatment and analysis. TSA results provide conditions in which the protein of interest is stable and therefore suitable to carry out further biophysical and structural characterization.

Asunto(s)

RESUMEN

Proteins from thermophilic organisms are a matter of immense interest for decades because of its application in fields like de-novo protein design, thermostable variants of biocatalysts etc. Previous studies have found several sequence and structural adaptations related to thermal stability, while charge reversal study remains ignored. Here we address whether charge reversal mutations naturally occur in mesophilic-thermophilic/hyperthermophilic orthologous proteins. Do they contribute to thermal stability? Our systematic study on 1550 mesophilic-thermophilic/hyperthermophilic orthologous protein pairs with remarkable structural and topological similarity, shows gain in coulombic interaction energy in thermophilic/hyperthermophilic proteins at short range associated with partially exposed and buried charge reversal mutations, which may enhance thermostability. Our findings call forth its application in future protein engineering studies. Communicated by Ramaswamy H. Sarma.

Asunto(s)

RESUMEN

Improving protein thermostability has been a labor- and time-consuming process in industrial applications of protein engineering. Advances in computational approaches have facilitated the development of more efficient strategies to allow the prioritization of stabilizing mutants. Among these is FEP+, a free energy perturbation implementation that uses a thoroughly tested physics-based method to achieve unparalleled accuracy in predicting changes in protein thermostability. To gauge the applicability of FEP+ to situations where crystal structures are unavailable, here we have applied the FEP+ approach to homology models of 12 different proteins covering 316 mutations. By comparing predictions obtained with homology models to those obtained using crystal structures, we have identified that local rather than global sequence conservation between target and template sequence is a determining factor in the accuracy of predictions. By excluding mutation sites with low local sequence identity (<40%) to a template structure, we have obtained predictions with comparable performance to crystal structures (R2 of 0.67 and 0.63 and an RMSE of 1.20 and 1.16 kcal/mol for crystal structure and homology model predictions, respectively) for identifying stabilizing mutations when incorporating residue scanning into a cascade screening strategy. Additionally, we identify and discuss inherent limitations in sequence alignments and homology modeling protocols that translate into the poor FEP+ performance of a few select examples. Overall, our retrospective study provides detailed guidelines for the application of the FEP+ approach using homology models for protein thermostability predictions, which will greatly extend this approach to studies that were previously limited by structure availability.

Asunto(s)

RESUMEN

Temperature is a fundamental environmental factor that shapes the evolution of organisms. Learning thermal determinants of protein sequences in evolution thus has profound significance for basic biology, drug discovery, and protein engineering. Here, we use a data set of over 3 million BRENDA enzymes labeled with optimal growth temperatures (OGTs) of their source organisms to train a deep neural network model (DeepET). The protein-temperature representations learned by DeepET provide a temperature-related statistical summary of protein sequences and capture structural properties that affect thermal stability. For prediction of enzyme optimal catalytic temperatures and protein melting temperatures via a transfer learning approach, our DeepET model outperforms classical regression models trained on rationally designed features and other deep-learning-based representations. DeepET thus holds promise for understanding enzyme thermal adaptation and guiding the engineering of thermostable enzymes.

Asunto(s)

RESUMEN

A number of scientific data indicate that γ-conglutin can be internalised by different human cells and undergoes secretion from the seed in response to high temperature. In both of these cases, the protein must interact in some manner with biological membranes, however, the mechanisms underlying this phenomenon remain unknown. Herein, we found that the remarkable change of total surface hydrophobicity after appropriate heat treatment of γ-conglutin monomer led to its interaction with model membranes (liposomes). Before the interaction, the protein undergoes an intriguing thermal unfolding pattern which was studied based on a spectroscopic approach. Insight into the interaction mechanism with liposomes was possible thanks to applying two molecular probes that were differentially localised in the lipid bilayer. The results show that the thermal rearranged γ-coglutin monomer affects hydrocarbon chains in model membranes leading to their morphology change and disruption. The main driving force of this phenomenon is based on hydrophobic interaction.

Asunto(s)

RESUMEN

The engineered three-helix bundle, UVF, is thermostabilized entropically due to heightened, native-state dynamics. However, it is unclear whether this thermostabilization strategy is observed in natural proteins from thermophiles. We performed all-atom, explicit solvent molecular dynamics simulations of two three-helix bundles from thermophilic H. butylicus (2lvsN and 2lvsC) and compared their dynamics to a mesophilic three-helix bundle, the Engrailed homeodomain (EnHD). Like UVF, 2lvsC had heightened native dynamics, which it maintained without unfolding at 100°C. Shortening and rigidification of loops in 2lvsN and 2lvsC and increased surface hydrogen bonds in 2lvsN were observed, as is common in thermophilic proteins. A buried disulfide and salt bridge in 2lvsN and 2lvsC, respectively, provided some stabilization, and addition of a homologous disulfide bond in EnHD slowed unfolding. The transferability and commonality of stabilization strategies among members of the three-helix bundle fold suggest that these strategies may be general and deployable in designing thermostable proteins.

Asunto(s)

RESUMEN

Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ1-pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.

Asunto(s)

RESUMEN

A multidimensional analysis aimed to determine the thermal impact on γ-conglutin at the two oligomeric states was carried out. A wide range of biophysical and bioinformatic methods allowed to get insight into a thermal unfolding mechanism. The determined midpoint transition temperature (Tm) values were remarkably different, being 56.5 °C and 71.1 °C for γ-conglutin monomer and hexamer, respectively. The unfolding pattern for hexamer molecules included aggregation/precipitation, while monomers tended to form soluble aggregates after heat exposure. Interestingly, differences in the aromatic amino acid residues movements indicate that during thermal treatment of γ-conglutin hexamer red-shift occurred contrary to the monomer in the case of which blue-shift was noted. The obtained results provide an essential contribution to expand our knowledge about the molecular characterization of this intriguing lupin seed protein.

Asunto(s)

RESUMEN

Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.

Asunto(s)

RESUMEN

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.

Asunto(s)

RESUMEN

Galdieria phlegrea is a polyextremophilic red alga belonging to Cyanidiophyceae. Galdieria phlegrea C-phycocyanin (GpPC), an abundant light-harvesting pigment with an important role in energy capture and transfer to photosystems, is the C-phycocyanin (C-PC) with the highest thermal stability described so far. GpPC also presents interesting antioxidant and anticancer activities. The X-ray structure of the protein was here solved. GpPC is a [(αß)3]2 hexamer, with the phycocyanobilin chromophore attached to Cys84α, Cys82ß and Cys153ß. Details of geometry and interaction with solvent of the chromophores are reported. Comparison with the structure of a C-PC in the entire Porphyridium purpureum phycobilisome system reveals that linker polypeptides have a significant effect on the local structure of the chromophores environment. Comparative analyses with the structures of other purified C-PCs, which were carried out including re-refined models of G. sulphuraria C-PC, reveal that GpPC presents a significantly higher number of inter-trimer salt bridges. Notably, the higher number of salt bridges at the (αß)3/(αß)3 interface is not due to an increased number of charged residues in this region, but to subtle conformational variations of their side chains, which are the result of mutations of close polar and non-polar residues.

Asunto(s)

RESUMEN

Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and resources used for protein purification and further studies. This protocol describes the use of the Thermofluor assay, in combination with a custom 24-condition screen, to identify buffer conditions that increase protein thermostability, using the conserved herpesviral protein UL37 as an example. Detailed instructions on screen conditions, running the Thermofluor MATLAB script, and analyzing the data are provided. In comparison to circular dichroism (CD), the buffer screen in combination with Thermofluor assay provides a faster and more informative method to analyze protein thermostability.

RESUMEN

Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe â†’ Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.

Asunto(s)