Factors determining the instability of the conformation of human mutant hemoglobins. A method for evaluating the instability

A method that allows to estimate the influence of single amino acid substitutions on the stability of proteins with known three-dimensional structure was developed. The role of 20 different physico-chemical properties of amino acids and some characteristics of the three-dimensional structure in the destabilization of protein structure caused by mutations was analyzed. Analysis of 54 proteins led us to reveal the characteristics of the three-dimensional structure. The parameters of the method were calculated on the basis of experimental data for 330 mutant human hemoglobins. The prediction accuracy of the method is on average 81%. The method may be used for the analysis of the total mutational spectra of the proteins and also for the estimation of effects of the single amino acid substitution on the protein structure in gene-engineering experiments.     

Human-readable rule generator for integrating amino scid sequence information and stability of mutant proteins

Most of the bioinformatics tools developed for predicting mutant protein stability appear as a black box and the relationship between amino acid sequence/structure and stability is hidden to the users. We have addressed this problem and developed a human-readable rule generator for integrating the knowledge of amino acid sequence and experimental stability change upon single mutation. Using information about the original residue, substituted residue, and three neighboring residues, classification rules have been generated to discriminate the stabilizing and destabilizing mutants and explore the basis for experimental data. These rules are human readable, and hence, the method enhances the synergy between expert knowledge and computational system. Furthermore, the performance of the rules has been assessed on a nonredundant data set of 1,859 mutants and we obtained an accuracy of 80 percent using cross validation. The results showed that the method could be effectively used as a tool for both knowledge discovery and predicting mutant protein stability. We have developed a Web for classification rule generator and it is freely available at http://bioinformatics.myweb.hinet.net/irobot.htm.     

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.     

Single amino acid substitutions producing instability of globular proteins. Calculation of their frequencies in the entire mutational spectra of the α- and β-subunits of human hemoglobin

Summary The frequencies of substitutions resulting in protein instability were calculated by a method estimating changes in stability produced by amino acid substitutions. The method takes into account the accessibility of an amino acid position to a solvent and changes in the specificity of amino acid interactions. When tested on human mutant hemoglobins, the method yielded predictions with a preciseness of 80%. The consideration of the evolutionary homologous proteins in the analysis allowed us to estimate the evolutionary constraints imposed on stability of their spatial structure. With these limitations, approximately 50% of amino acid substitutions in the entire mutational spectra of the - and -subunits of human hemoglobin were found to damage the spatial structure of the globular proteins.     

Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

Prediction of the effect of amino acid substitutions on the thermodynamic stability of proteins is of great importance for studies into the molecular mechanisms underlying the abnormal function of mutant proteins, interpretation of genotyping results, and purposeful design of modified proteins with improved biomedical and biotechnological properties. A set of methods was developed for predicting the changes in free energy (ΔΔG) of mutant proteins containing single substitutions using the information only about protein primary structure or also about the spatial structure. A modified KRAB algorithm was used; its higher accuracy in predicting the changes in the thermodynamic stability of mutant proteins compared with the other known methods designed for solving this problem is demonstrated. Distribution of the positions in the sequence of Malayan pit viper venom protein (kistrin) where the substitutions decrease or increase kistrin stability is analyzed. The substitutions at most positions conserved in the disintegrin family decrease the stability of this protein, except for several positions whose conservation can be determined by functional significance.     

Estimation of the frequency of amino acid substitutions causing instability of the spatial structure in an overall mutation spectrum of alpha- and beta-subunits of human hemoglobin

The influence of single amino acid substitutions on the stability of alpha- and beta-chains of human hemoglobin was investigated by the computer method. The method was based on characteristics of protein tertiary structure and physico-chemical properties of amino acids. Frequencies of unstable mutations in the total mutational spectra of alpha- and beta-subunits of human hemoglobin were analysed: instability was produced by 26% of mutations in the alpha-subunit and by 32% in the beta-subunit. These results support the idea that certain limitations exist for stability changes produced by amino acid substitutions.     

C-terminal region of protein kinase CK2 alpha: How the structure can affect function and stability of the catalytic subunit

A novel mutant of the catalytic alpha subunit of human protein kinase CK2 (CK2 alpha) was designed in an attempt to clarify the role of the carboxylic-terminal segment characteristic of vertebrates, excluding fish. Starting from the sequence alignments, we constructed a phylogenetic tree of the primary structure of CK2 alpha. On this basis, we substituted two distal prolines with alanines (PA 382-384). Theoretical calculations and spectropolarimetry measurements, performed both on native and mutant subunits, indicate an increased content of alpha-helix after this double amino acidic substitution. In order to clarify the structure/function relationship of the C-terminal region, we verified if the structural change affects the catalytic activity of CK2 alpha. The mutant exhibits slightly increased phosphorylation efficiency, but reduced ability to transfer phosphate in comparison with the native subunit. At last, we compared the thermal stability of the mutant with respect to the native subunit and we tested the proteolytic degradability. The observation that the PA 382-384 mutant exhibits an increased thermal and proteolytic stability suggests that this mutant could be employed to solve the three-dimensional (3D) structure of human CK2 alpha and to overcome difficulties in crystallizing the native form.     

Role of amino acid residues at turns in the conformational stability and folding of human lysozyme

To clarify the role of amino acid residues at turns in the conformational stability and folding of a globular protein, six mutant human lysozymes deleted or substituted at turn structures were investigated by calorimetry, GuHCl denaturation experiments, and X-ray crystal analysis. The thermodynamic properties of the mutant and wild-type human lysozymes were compared and discussed on the basis of their three-dimensional structures. For the deletion mutants, Delta47-48 and Delta101, the deleted residues are in turns on the surface and are absent in human alpha-lactalbumin, which is homologous to human lysozyme in amino acid sequence and tertiary structure. The stability of both mutants would be expected to increase due to a decrease in conformational entropy in the denatured state; however, both proteins were destabilized. The destabilizations were mainly caused by the disappearance of intramolecular hydrogen bonds. Each part deleted was recovered by the turn region like the alpha-lactalbumin structure, but there were differences in the main-chain conformation of the turn between each deletion mutant and alpha-lactalbumin even if the loop length was the same. For the point mutants, R50G, Q58G, H78G, and G37Q, the main-chain conformations of these substitution residues located in turns adopt a left-handed helical region in the wild-type structure. It is thought that the left-handed non-Gly residue has unfavorable conformational energy compared to the left-handed Gly residue. Q58G was stabilized, but the others had little effect on the stability. The structural analysis revealed that the turns could rearrange the main-chain conformation to accommodate the left-handed non-Gly residues. The present results indicate that turn structures are able to change their main-chain conformations, depending upon the side-chain features of amino acid residues on the turns. Furthermore, stopped-flow GuHCl denaturation experiments on the six mutants were performed. The effects of mutations on unfolding-refolding kinetics were significantly different among the mutant proteins. The deletion/substitutions in turns located in the alpha-domain of human lysozyme affected the refolding rate, indicating the contribution of turn structures to the folding of a globular protein.     

Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree

We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.     

Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships

Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus. However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, respectively) to the C-terminal region of the truncated enzyme. Three structures of these mutant enzymes were determined by an X-ray diffraction method. All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model. Thermal stability of these mutant enzymes is discussed based on the 3D structure with special attention to the width of the active-site groove and the minor groove, distortion of beta-sheet pillar structure and size of cavity in the domain-domain interface around the C-terminus. Our previous studies revealed that the thermal stability of isopropylmalate dehydrogenase increases when the active-site cleft is closed (the closed form). In the present study it is shown that the active-site cleft can be regulated by open-close movement of the minor groove located at the opposite side to the active-site groove on the same subunit, through a paperclip-like motion.     

Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412–1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89–905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124–160 and 341–368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1''s amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function.     

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.     

Prediction of protein mutant stability using classification and regression tool

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.     

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.     

iPTREE-STAB: interpretable decision tree based method for predicting protein stability changes upon mutations

We have developed a web server, iPTREE-STAB for discriminating the stability of proteins (stabilizing or destabilizing) and predicting their stability changes (delta deltaG) upon single amino acid substitutions from amino acid sequence. The discrimination and prediction are mainly based on decision tree coupled with adaptive boosting algorithm, and classification and regression tree, respectively, using three neighboring residues of the mutant site along N- and C-terminals. Our method showed an accuracy of 82% for discriminating the stabilizing and destabilizing mutants, and a correlation of 0.70 for predicting protein stability changes upon mutations. AVAILABILITY: http://bioinformatics.myweb.hinet.net/iptree.htm. SUPPLEMENTARY INFORMATION: Dataset and other details are given.     

Role of surface hydrophobic residues in the conformational stability of human lysozyme at three different positions

To evaluate the contribution of the amino acid residues on the surface of a protein to its stability, a series of hydrophobic mutant human lysozymes (Val to Gly, Ala, Leu, Ile, Met, and Phe) modified at three different positions on the surface, which are located in the alpha-helix (Val 110), the beta-sheet (Val 2), and the loop (Val 74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by X-ray crystallography at 100 K, respectively. Differences in the denaturation Gibbs energy change between the wild-type and the hydrophobic mutant proteins ranged from 4.6 to -9.6 kJ/mol, 2.7 to -1.5 kJ/mol, and 3.6 to -0.2 kJ/mol at positions 2, 74, and 110, respectively. The identical substitution at different positions and different substitutions at the same position resulted in different degrees of stabilization. Changes in the stability of the mutant proteins could be evaluated by a unique equation considering the conformational changes due to the substitutions [Funahashi et al. (1999) Protein Eng. 12, 841-850]. For this calculation, secondary structural propensities were newly considered. However, some mutant proteins were not adapted to the equation. The hydration structures around the mutation sites of the exceptional mutant proteins were affected due to the substitutions. The stability changes in the exceptional mutant proteins could be explained by the formation or destruction of the hydration structures. These results suggest that the hydration structure mediated via hydrogen bonds covering the protein surface plays an important role in the conformational stability of the protein.     

Role of the C-terminal tail on the quaternary structure of aldehyde dehydrogenases

To assess the importance of the C-terminal tail in the structure of aldehyde dehydrogenases (ALDH), mutants of tetrameric ALDH1 were generated by adding a tail of 5 amino acids (ALDH1-5aa) or the tail from the class 3 enzyme. A mutant of dimeric ALDH3 was made, where 17 amino acids from the C-terminus were deleted to generate ALDH3DeltaTail. The expression and solubility of the ALDH1 mutants was slightly lower than the wild type. Expression of ALDH3DeltaTail mutant was similar to wild type, but the solubility was only about 30%. The activity of ALDH1-5aa mutant was 30%, while ALDH1-H3Tail mutant was 60% active, compared to the wild type. The activity of the class 3 mutant was similar to the activity of the parent ALDH3 enzyme. Analysis of stability against temperature demonstrated that ALDH1-5aa was more stable than ALDH1 wild type, while the ALDH1-H3Tail mutant was considerably less stable than ALDH1, showing a stability similar to ALDH3. However, native gel and size exclusion analysis, showed no changes in the oligomerization state of these mutants. ALDH3DeltaTail mutant was more stable than wild type; the stability against temperature was similar to ALDH1. The ALDH3DeltaTail mutant showed an elution similar to that of ALDH1 from the size exclusion column, indicating that it was possibly a tetramer. These results show that the tail in ALDH3, is involved in the determination of the quaternary structure of ALDH3, but has no effect on the ALDH1 enzyme; the absence of the C-terminal tail is not the only factor participating in holding the dimers together. Thus, the interaction between single residues, or interactions with the N-terminal region might be more important for maintaining stable tetramers.     

Role of the C-terminal tail on the quaternary structure of aldehyde dehydrogenases

To assess the importance of the C-terminal tail in the structure of aldehyde dehydrogenases (ALDH), mutants of tetrameric ALDH1 were generated by adding a tail of 5 amino acids (ALDH1-5aa) or the tail from the class 3 enzyme. A mutant of dimeric ALDH3 was made, where 17 amino acids from the C-terminus were deleted to generate ALDH3ΔTail. The expression and solubility of the ALDH1 mutants was slightly lower than the wild type. Expression of ALDH3ΔTail mutant was similar to wild type, but the solubility was only about 30%. The activity of ALDH1-5aa mutant was 30%, while ALDH1-H3Tail mutant was 60% active, compared to the wild type. The activity of the class 3 mutant was similar to the activity of the parent ALDH3 enzyme. Analysis of stability against temperature demonstrated that ALDH1-5aa was more stable than ALDH1 wild type, while the ALDH1-H3Tail mutant was considerably less stable than ALDH1, showing a stability similar to ALDH3. However, native gel and size exclusion analysis, showed no changes in the oligomerization state of these mutants. ALDH3ΔTail mutant was more stable than wild type; the stability against temperature was similar to ALDH1. The ALDH3ΔTail mutant showed an elution similar to that of ALDH1 from the size exclusion column, indicating that it was possibly a tetramer. These results show that the tail in ALDH3, is involved in the determination of the quaternary structure of ALDH3, but has no effect on the ALDH1 enzyme; the absence of the C-terminal tail is not the only factor participating in holding the dimers together. Thus, the interaction between single residues, or interactions with the N-terminal region might be more important for maintaining stable tetramers.     

Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart

Mesophilic cytochrome c(551) of Pseudomonas aeruginosa (PA c(551)) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c(552) through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c(551) could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c(552), there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.