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) estimatesthe changes in conformational stability due to single aminoacid substitutions using a pseudo-energy potential developedfor evaluating structure–sequence compatibility in thestructure prediction method, the 3D–1D compatibility evaluation.Nine mutant human lysozymes expected to significantly increasein stability from SPMP were constructed, in order to experimentallyverify the reliability of SPMP. The thermodynamic parametersfor denaturation and crystal structures of these mutant proteinswere determined. One mutant protein was stabilized as expected,compared with the wild-type protein. However, the others werenot stabilized even though the structural changes were subtle,indicating that SPMP overestimates the increase in stabilityor underestimates negative effects due to substitution. Thestability changes in the other mutant human lysozymes previouslyreported were also analyzed by SPMP. The correlation of thestability changes between the experiment and prediction dependedon the types of substitution: there were some correlations forproline mutants and cavity-creating mutants, but no correlationfor mutants related to side-chain hydrogen bonds. The presentresults may indicate some additional factors that should beconsidered in the calculation of SPMP, suggesting that SPMPcan be refined further.     

Improved conformational space annealing method to treat β-structure with the UNRES force-field and to enhance scalability of parallel implementation

Successful application of physics-based protein-structure prediction methods depends on sophisticated computational approaches to global optimization of the conformational energy of a polypeptide chain. One of the most effective procedures for the global optimization of protein structures appears to be the Conformational Space Annealing (CSA) method. CSA is a hybrid method which combines genetic algorithms, essential aspects of the build-up method and a local gradient-based minimization. CSA evolves the population of conformations through genetic operators (mutations, i.e. perturbations of selected geometric parameters, and crossovers, i.e. exchange of selected subsets of geometric parameters between conformations) to a final population optimizing their conformational energy. Implementation of the CSA method with the united-residue force field (UNRES, in which each amino-acid residue is represented by two interaction sites, namely the united peptide group and the united side-chain) was enhanced by introducing new crossover operations consisting of (i) copying β-hairpins, (ii) copying remote strand pairs forming non-local β-sheets, and (iii) copying α-helical segments. A mutation operation, which shifts the position of a β-turn, was also introduced. The new operations promote β-structure, and are essential for searching the conformational space of proteins containing both α- and β-structure; without these operations, excessive preference of α-helical structures is obtained, even though these structures are high in energy. Parallelization of the CSA method has also been enhanced by removing most of the synchronization steps; the improved algorithm scales almost linearly up to 1,000 processors with over 75% average performance.     

A study of structural determinants in the interleukin-1 fold

The structures of interleukin-1ß, basic fibroblastgrowth factor and Erythrina trypsin inhibitor have been analysedin order to determine whether the hydrophobic core remains conserved,even when the structures have extremely low sequence similarities.We find that there are significant differences in the way eachprotein achieves a satisfactory arrangement of core residuesand that positions which contribute to the core of one structureare not guaranteed to contribute to the integrity of another.Furthermore, the side-chain packing arrangements of these coreresidues vary significantly between the three structures. Duringthis analysis the side-chain rotamers for three independentlydetermined interleukin-1ß structures were also compared.It was found that although buried residues are generally inagreement the remaining residues frequently occupy differentrotamers in the three structures. This suggests that althoughmeaningful studies are possible for buried side-chains the resultsobtained from equivalent analyses of accessible residues shouldbe treated with caution. These results are discussed with specificreference to the optimization of side-chain packing in proteinsof known structure.     

Protein sequence entropy is closely related to packing density and hydrophobicity

We investigated the correlation between the Shannon information entropy, 'sequence entropy', with respect to the local flexibility of native globular proteins as described by inverse packing density. These are determined at each residue position for a total set of 130 query proteins, where sequence entropies are calculated from each set of aligned residues. For the accompanying aggregate set of 130 alignments, a strong linear correlation is observed between the calculated sequence entropy and the corresponding inverse packing density determined at an associated residue position. This region of linearity spans the range of C(alpha) packing densities from 12 to 25 amino acids within a sphere of 9 angstrom radius. Three different hydrophobicity scales all mimic the behavior of the sequence entropies. This confirms the idea that the ability to accommodate mutations is strongly dependent on the available space and on the propensity for each amino acid type to be buried. Future applications of these types of methods may prove useful in identifying both core and flexible residues within a protein.     

Analysis and prediction of inter-strand packing distances between {beta}-sheets of globular proteins

Any two ß-strands belonging to two different ß-sheetsin a protein structure are considered to pack interactivelyif each ß-strand has at least one residue that undergoesa loss of one tenth or more of its solvent contact surface areaupon packing. A data set of protein 3-D structures (determinedat 2.5 Å resolution or better), corresponding to 428 proteinchains, contains 1986 non-identical pairs of ß-strandsinvolved in interactive packing. The inter-axial distance betweenthese is significantly correlated to the weighted sum of thevolumes of the interacting residues at the packing interface.This correlation can be used to predict the changes in the inter-sheetdistances in equivalent ß-sheets in homologous proteinsand, therefore, is of value in comparative modelling of proteins.     

Knowledge-based potential defined for a rotamer library to design protein sequences

A knowledge-based potential for a rotamer library was developedto design protein sequences. Protein side-chain conformationsare represented by 56 templates. Each of their fitness to agiven structural site-environment is evaluated by a combinedfunction of the three knowledge-based terms, i.e. two-body side-chainpacking, one-body hydration and local conformation. The numberof matches between the native sequence and the structural site-environmentin the database and that of the virtually settled mismatches,counted in advance, were transformed into the energy scores.In the best-14 test (assessment for the reproduction abilityof the native rotamer on its structural site within a quarterof 56 fitness rank positions), the structural stability analysison mutants of human and T4 lysozymes and the inverse-foldingsearch by a structure profile against the sequence database,this function performs better than the function deduced withthe conventional normalization and our previously developedfunction. Targeting various structural motifs, de novo sequencedesign was conducted with the function. The sequences thus obtainedexhibit reasonable molecular masses and hydrophobic/hydrophilicpatterns similar to the native sequences of the target and actas if they were the homologs to the target proteins in BLASTPsearch. This significant improvement is discussed in terms ofthe reference state for normalization and the crucial role ofshort-range repulsion to prohibit residue bumps.     

Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction

De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction.     

从浓度分布评价填料塔性能

精馏塔性能的优劣通常可以用在塔顶和塔底取样,然后计算其分离效率的方法来判断,但这种方法只可以判断出精馏设备效率的高低,却几乎无法找出造成设备效率不够理想或低效的原因．不良初始分布、壁流、沟流和溪流、端效应以及气相或液相返混等现象都可以造成填料塔效率的下降,而     

Prediction of the structures of proteins with the UNRES force field, including dynamic formation and breaking of disulfide bonds

The presence of disulfide bonds is essential for maintaining the structure and function of many proteins. The disulfide bonds are usually formed dynamically during folding. This process is not accounted for in present algorithms for protein-structure prediction, which either deduce the possible positions of disulfide bonds only after the structure is formed or assume fixed disulfide bonds during the course of simulated folding. In this work, the conformational space annealing (CSA) method and the UNRES united-residue force field were extended to treat dynamic formation of disulfide bonds. A harmonic potential is imposed on the distance between disulfide-bonded cysteine side-chain centroids to describe the energetics of bond distortion and an energy gain of 5.5 kcal/mol is added for disulfide-bond formation. Formation, breaking and rearrangement of disulfide bonds are included in the CSA search by introducing appropriate operations; the search can also be carried out with a fixed disulfide-bond arrangement. The algorithm was applied to four proteins: 1EI0 (alpha), 1NKL (alpha), 1L1I (beta-helix) and 1ED0 (alpha + beta). For 1EI0, a low-energy structure with correct fold was obtained both in the runs without and with disulfide bonds; however, it was obtained as the lowest in energy only with the native disulfide-bond arrangement. For the other proteins studied, structures with the correct fold were obtained as the lowest (1NKL and 1L1I) or low-energy structures (1ED0) only in runs with disulfide bonds, although the final disulfide-bond arrangement was non-native. The results demonstrate that, by including the possibility of formation of disulfide bonds, the predictive power of the UNRES force field is enhanced, even though the disulfide-bond potential introduced here rarely produces disulfide bonds in native positions. To the best of our knowledge, this is the first algorithm for energy-based prediction of the structure of disulfide-bonded proteins without any assumption as to the positions of native disulfides or human intervention. Directions for improving the potentials and the search method are suggested.     

The Equilibrium Principle of Displacement Chromatography

Abstract

Displacement chromatography, an alternate method to elution chromatography in terms of operation, is described. The principle of displacement is conveniently explained using the concept of an ‘addend’ which is regarded as a species essential to any equilibrium reaction consisting of association of two species and its reverse reaction. Microscopic equilibria in a chromatographic column can be characterized by use of reduction potential strengths (Δμ° and Δμ) and its derived quantities, S and L potentials that we introduced for expression of equilibria. The method for evaluating the separation factor of displacement chromatography is also described. Further, a profile of simulated separation produced by iterative applications of the distribution function to the multistage equilibrium in the column is presented.     

Computational analysis of alpha-helical membrane protein structure: implications for the prediction of 3D structural models

Relatively little has been known about the structure of alpha-helical membrane proteins, since until recently few structures had been crystallized. These limited data have restricted structural analyses to the prediction of secondary structure, rather than tertiary folds. In order to address this, this paper describes an analysis of the 23 available membrane protein structures. A number of findings are made that are of particular relevance to transmembrane helix packing: (1) on average lipid-tail-accessible transmembrane residues are significantly more hydrophobic, less conserved and contain different residue types to buried residues; (2) charged residues are not always buried and, when accessible to membrane lipid tails, few are paired with another charge and instead they often interact with phospholipid head-groups or with other residue types; (3) a significant proportion of lipid-tail-accessible charged and polar residues form hydrogen bonds only with residues one turn away in the same helix (intra-helix); (4) pore-lining residues are usually hydrophobic and it is difficult to distinguish them from buried residues in terms of either residue type or conservation; and (5) information was gained about the proportion of helices that tend to contribute to lining a pore and the resulting pore diameter. These findings are discussed with relevance to the prediction of membrane protein 3D structure.     

Predicting drug metabolism by cytochrome P450 2C9: comparison with the 2D6 and 3A4 isoforms

By the use of knowledge gained through modeling of drug metabolism mediated by the cytochrome P450 2D6 and 3A4 isoforms, we constructed a 2D-based model for site-of-metabolism prediction for the cytochrome P450 2C9 isoform. The similarities and differences between the models for the 2C9 and 2D6 isoforms are discussed through structural knowledge from the X-ray crystal structures and trends in experimental data. The final model was validated on an independent test set, resulting in an area under the curve value of 0.92, and a site of metabolism was found among the top two ranked atoms for 77% of the compounds.     

苯甲酸釜残液全部回收的工艺开发利用

Benzoic acid residue is solid waste produced from the production of benzoic acid by oxidizing toluene. Because it contained important chemical raw materials such as benzoic acid, benzyl benzoate and fluorenone, it is necessary to recover them from the residue. In this work the technique featured with high efficiency evaporation and vacuum distillation was developed to obtain total recovery and utilization of the benzoic acid residue. By controlling the operation temperature at 260°C and the pressure of 16 kPa in the rising and falling film evaporators, heavy components separated efficiently from the residue can be polymerized and the light components consisting of 63% of the residue entered into a benzoic acid vacuum distillation column. Keeping the temperature of polymerization at (280±10)°C, coumarone resin was produced after adjusting the softening point according to the market re-quirements. Vacuum distillation was operated under the following conditions: top temperature at 186°C, top pres-sure of 16 kPa, bottom temperature at 240-250°C, reflux ratio being 3︰1. Benzoic acid of 98% purity was distilled out from the column as a side stream and the bottom product was crude benzyl benzoate. By the developed tech-nique, the benzoic acid residue was splitted into three products, benzoic acid, crude benzyl benzoate and coumarone resin without any surplus waste.     

A mutation at the interface between domains causes rearrangement of domains in 3-isopropylmalate dehydrogenase

The structure of a thermostable Ala172Leu mutant, designated A172L, of 3-isopropylmalate dehydrogenase from Thermus thermophilus was determined. The crystal belongs to space group P2(1), with cell parameters a = 55.5 A, b = 88.1 A, c = 72.0 A and beta = 100.9 degrees. There is one dimer in each asymmetric unit. The final R factor is 17.8% with 69 water molecules at 2.35 A resolution. The mutation is located at the interface between domains and the C alpha trace of the mutant structure deviates from that of the native structure by as much as 1.7 A, while the structure of each domain barely changes. The mutant enzyme has a more closed conformation compared with the wild-type enzyme as a result of the replacement of Ala with Leu at residue 172. These structural variations were found independent of the crystal packing, because the structure of wild type was the same in crystals obtained in different precipitants. The hinge regions for the movement of domains are located around the active cleft of the enzyme, an observation that implies that the mobility of domains around the hinge is indispensable for the activity of the enzyme. The larger side chain at the mutated site contributed to the thermostability of the mutant protein by enhancing the local packing of side chains, and also by shifting the backbone of the opposing domain.      

Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY

The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57. However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses. Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage. Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees C. Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.      

Twin-screw extrusion technology,an original solution for the extraction of proteins from alfalfa (Medicago sativa)

The aim of this study is to develop a new approach to green plant fractionation using twin-screw extrusion, leading to the production of a green filtrate, rich in proteins and a solid fibrous residue, rich in cellulose. The influence of the screw profile on protein recovery in the liquid extract and on plant dehydration was characterized. Two screw profiles were tested, at different temperatures and liquid/solid ratios. Increasing the shearing action on the plant produces a liquid extract richer in protein; from 25% of dry matter to 31% of dry matter when two additional sections of paddle screw elements are inserted onto the screw profile. However, it leads to higher liquid retention in the alfalfa fibers and thus dehydration of the plant is less efficient. The fibrous residue still contains at least 60% humidity when the alfalfa is highly degraded, whereas less than 47% humidity remain with only one section of bilobe paddle screws on the profile. For the two screw profiles tested, the crucial parameter influencing the fraction quality (protein content of the filtrate, dry matter content of the fibrous residue) is the liquid/solid ratio.     

Optimizing potentials for the inverse protein folding problem

Inverse protein folding, which seeks to identify sequences that fold into a given structure, has been approached by threading candidate sequences onto the structure and scoring them with database-derived potentials. The sequences with the lowest energies are predicted to fold into that structure. It has been argued that the limited success of this type of approach is not due to the discrepancy between the scoring potential and the true potential but is rather due to the fact that sequences choose their lowest-energy structure rather than structures choosing the lowest-energy sequences. Here we develop a non- physical potential scheme optimized for the inverse folding problem. We maximize the average probability of success for a set of lattice proteins to obtain the optimal potential energy function, and show that the potential obtained by our method is more likely to produce successful predictions than the true potential.      

Synthesis and characterization of phosphonate and aromatic-based polynorbornene polymers derived from the ring opening metathesis polymerization method and investigation of their thermal properties

In the present study, phosphonate ester, phosphonic acid, and aromatic (phenyl, naphthalene, anthracene) groups containing polymers were synthesized by the ROMP method to analyze thermal properties of these polymers. Thermal stability of the synthesized polymers is tested by thermal gravimetric analysis under nitrogen, air, and microscale combustion calorimetry analysis. Analysis shows that thermal behavior is directly related to the phosphorus level in the copolymer series. All the polymers are thermally stable under nitrogen and air up to 900 °C. Synergistic charring effect under air was observed between aromatic groups and phosphonic acid functionality in the copolymer series. Anthracene units have a greater potential to form carbonaceous char than the naphthalene and phenyl units. Phosphonate ester and naphthalene units bearing copolymers (P3A) gave 8.13% char yield at 900 °C under air. Phosphonic acid derivatives of this polymer, P3D, gave a highest char residue of 17.15% under the same condition. The introduction of phosphonate and phosphonic acid in each copolymer series is also beneficial in reducing the peak heat release rate (PHRR). Cleavage of the phosphonate ester bearing homopolymer (P4) to phosphonic acid (P4A) causes a sharp decrease in the PHRR ratio from 274 to 28.2 W/g. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47085.     

Grow to Fit Molecular Dynamics (G2FMD): an ab initio method for protein side-chain assignment and refinement

The rough energy landscapes and tight packing of protein interiors are two of the critical factors that have prevented the wide application of physics-based models in protein side-chain assignment and protein structure prediction in general. Complementing the rotamer-based methods, we propose an ab initio method that utilizes molecular mechanics simulations for protein side-chain assignment and refinement. By reducing the side-chain size, a smooth energy landscape was obtained owing to the increased distances between the side chains. The side chains then gradually grow back during molecular dynamics simulations while adjusting to their surrounding driven by the interaction energies. The method overcomes the barriers due to tight packing that limit conformational sampling of physics-based models. A key feature of this approach is that the resulting structures are free from steric collisions and allow the application of all-atom models in the subsequent refinement. Tests on a small set of proteins showed nearly 100% accuracy on both chi1 and chi2 of buried residues and 94% of them were within 20 degrees from the native conformation, 79% were within 10 degrees and 42% were within 5 degrees . However, the accuracy decreased when exposed side chains were involved. Further improvement and application of the method and the possible reasons that affect the accuracy on the exposed side chains are discussed.