FireProt 2.0: web-based platform for the fully automated design of thermostable proteins.

Thermostable proteins find their use in numerous biomedical and biotechnological applications. However, the computational design of stable proteins often results in single-point mutations with a limited effect on protein stability. However, the construction of stable multiple-point mutants can prove difficult due to the possibility of antagonistic effects between individual mutations. FireProt protocol enables the automated computational design of highly stable multiple-point mutants. FireProt 2.0 builds on top of the previously published FireProt web, retaining the original functionality and expanding it with several new stabilization strategies. FireProt 2.0 integrates the AlphaFold database and the homology modeling for structure prediction, enabling calculations starting from a sequence. Multiple-point designs are constructed using the Bron-Kerbosch algorithm minimizing the antagonistic effect between the individual mutations. Users can newly limit the FireProt calculation to a set of user-defined mutations, run a saturation mutagenesis of the whole protein or select rigidifying mutations based on B-factors. Evolution-based back-to-consensus strategy is complemented by ancestral sequence reconstruction. FireProt 2.0 is significantly faster and a reworked graphical user interface broadens the tool's availability even to users with older hardware. FireProt 2.0 is freely available at http://loschmidt.chemi.muni.cz/fireprotweb.

Predicting DNA-binding protein and coronavirus protein flexibility using protein dihedral angle and sequence feature.

The flexibility of protein structure is related to various biological processes, such as molecular recognition, allosteric regulation, catalytic activity, and protein stability. At the molecular level, protein dynamics and flexibility are important factors to understand protein function. DNA-binding proteins and Coronavirus proteins are of great concern and relatively unique proteins. However, exploring the flexibility of DNA-binding proteins and Coronavirus proteins through experiments or calculations is a difficult process. Since protein dihedral rotational motion can be used to predict protein structural changes, it provides key information about protein local conformation. Therefore, this paper introduces a method to improve the accuracy of protein flexibility prediction, DihProFle (Prediction of DNA-binding proteins and Coronavirus proteins flexibility introduces the calculated dihedral Angle information). Based on protein dihedral Angle information, protein evolution information, and amino acid physical and chemical properties, DihProFle realizes the prediction of protein flexibility in two cases on DNA-binding proteins and Coronavirus proteins, and assigns flexibility class to each protein sequence position. In this study, compared with the flexible prediction using sequence evolution information, and physicochemical properties of amino acids, the flexible prediction accuracy based on protein dihedral Angle information, sequence evolution information and physicochemical properties of amino acids improved by 2.2% and 3.1% in the nonstrict and strict conditions, respectively. And DihProFle achieves better performance than previous methods for protein flexibility analysis. In addition, we further analyzed the correlation of amino acid properties and protein dihedral angles with residues flexibility. The results show that the charged hydrophilic residues have higher proportion in the flexible region, and the rigid region tends to be in the angular range of the protein dihedral angle (such as the ψ angle of amino acid residues is more flexible than rigid in the range of 91°-120°). Therefore, the results indicate that hydrophilic residues and protein dihedral angle information play an important role in protein flexibility.

The structure of protein dynamic space.

We use a bioinformatic description of amino acid dynamic properties, based on residue-specific average B factors, to construct a dynamics-based, large-scale description of a space of protein sequences. We examine the relationship between that space and an independently constructed, structure-based space comprising the same sequences. It is demonstrated that structure and dynamics are only moderately correlated. It is further shown that helical proteins fall into two classes with very different structure-dynamics relationships. We suggest that dynamics in the two helical classes are dominated by distinctly different modes--pseudo-one-dimensional, localized helical modes in one case, and pseudo-three-dimensional (3D) global modes in the other. Sheet/barrel and mixed-α/ß proteins exhibit more conventional structure-dynamics relationships. It is found that the strongest correlation between structure and dynamic properties arises when the latter are represented by the sequence average of the dynamic index, which corresponds physically to the overall mobility of the protein. None of these results are accessible to bioinformatic methods hitherto available.

Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions.

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24-30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

Activation/Inhibition of Cholinesterases by Excess Substrate: Interpretation of the Phenomenological b Factor in Steady-State Rate Equation.

Cholinesterases (ChEs) display a non-michaelian behavior with positively charged substrates. In the steady-state rate equation, the b factor describes this behavior: if b > 1 there is substrate activation, if b < 1 there is substrate inhibition. The mechanistic significance of the b factor was investigated to determine whether this behavior depends on acylation, deacylation or on both steps. Kinetics of human acetyl- (AChE) and butyryl-cholinesterase (BChE) were performed under steady-state conditions and using a time-course of complete substrate hydrolysis. For the hydrolysis of short acyl(thio)esters, where acylation and deacylation are partly rate-limiting, steady-state kinetic analysis could not decide which step determines b. However, the study of the hydrolysis of an arylacylamide, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), where acetylation is rate-limiting, showed that b depends on the acylation step. The magnitude of b and opposite b values between AChE and BChE for the hydrolysis of acetyl(thio)- versus benzoyl-(thio) esters, then indicated that the productive adjustment of substrates in the active center at high concentration depends on motions of both the Ω and the acyl-binding loops. Benzoylcholine was shown to be a poor substrate of AChE, and steady-state kinetics showed a sudden inhibition at high concentration, likely due to the non-dissociation of hydrolysis products. The poor catalytic hydrolysis of this bulky ester by AChE illustrates the importance of the fine adjustment of substrate acyl moiety in the acyl-binding pocket. Molecular modeling and QM/MM simulations should definitively provide evidence for this statement.

The reproducible normality of the crystallographic B-factor.

Reproducibility determines the utility of a measurement. In structural biology the reproducibility permeate areas such as mechanics, data measurement, data analysis and refinement. In order to access the reproducibility of the combined contribution of these sources in uncertainties of protein crystallography we evaluated four groups of parameters from data collection to final structural model. We used lysozyme as a model, with 20 datasets collected at 1.6 Å resolution using two dissimilar x-ray diffraction setups and refined through a single automatic pipeline without arbitrary interpretation. Besides statistical differences in some structural parameters, the reproducibility of the final refined models allowed the determination of positional uncertainty, in good agreement with the Luzzati coordinate error. While the raw B-factor was found non-reproducible, an empirical scaling/normalization resulted in reproducible B-factors. The validity of this empirical scaling was corroborated by the reproducibility of normalized B-factors of independently solved datasets from proteins (insulin and myoglobin) from varying space groups available from structural database. The reproducibility of normalized B-factor may reposition this displacement parameter in the analysis of chemical (ligands, pH) and physical (pressure, temperature, space groups) variables.

Screening and identification of phytochemical drug molecules against mutant BRCA1 receptor of breast cancer using computational approaches.

The American Cancer Society claims that breast cancer is the second most significant cause of cancer-related death, with over one million women diagnosed each year. Breast cancer linked to the BRCA1 gene has a significant risk of mortality and recurrence and is susceptible to alteration or over-expression, which can lead to hereditary breast cancer. Given the shortage of effective and possibly curative treatments for breast cancer, the present study combined molecular and computational analysis to find prospective phytochemical substances that can suppress the mutant gene (BRCA1) that causes the disease. Virtual screening and Molecular docking approaches are utilized to find probable phytochemicals from the ZINC database. The 3D structure of mutant BRCA1 protein with the id 3PXB was extracted from the NCBI-PDB. Top 10 phytochemical compounds shortlisted based on molecular docking score between - 11.6 and - 13.0. Following the ADMET properties, only three (ZINC000085490903 = - 12.50, ZINC000085490832 = - 12.44, and ZINC000070454071 = - 11.681) of the 10 selected compounds have drug-like properties. The molecular dynamic simulation study of the top three potential phytochemicals showed stabilized RMSD and RMSF values as compared to the APO form of the BRCA1 receptor. Further, trajectory analysis revealed that approximately similar radius of gyration score tends to the compactness of complex structure, and principal component and cross-correlation analysis suggest that the residues move in a strong correlation. Thermostability of the target complex (B-factor) provides information on the stable energy minimized structure. The findings suggest that the top three ligands show potential as breast cancer inhibitors.

Decline of protein structure rigidity with interatomic distance.

BACKGROUND: Protein structural rigidity was analyzed in a non-redundant ensemble of high-resolution protein crystal structures by means of the Hirshfeld test, according to which the components (uX and uY) of the B-factors of two atoms (X and Y) along the interatomic direction is related to their degree of rigidity: the atoms may move as a rigid body if uX = uY and they cannot if uX ≠ uY. RESULTS: It was observed that the rigidity degree diminishes if the number of covalent bonds intercalated between the two atoms (d_seq) increases, while it is rather independent on the Euclidean distance between the two atoms (d): for a given value of d_seq, the difference between uX and uY does not depend on d. No additional rigidity decline is observed when d_seq ≥ ~ 30 and this upper limit is very modest, close to 0.015 Å. CONCLUSIONS: This suggests that protein flexibility is not fully described by B-factors that capture only partially the wide range of distortions that proteins can afford.

The amounts of thermal vibrations and static disorder in protein X-ray crystallographic B-factors.

Crystallographic B-factors provide direct dynamical information on the internal mobility of proteins that is closely linked to function, and are also widely used as a benchmark in assessing elastic network models. A significant question in the field is: what is the exact amount of thermal vibrations in protein crystallographic B-factors? This work sets out to answer this question. First, we carry out a thorough, statistically sound analysis of crystallographic B-factors of over 10 000 structures. Second, by employing a highly accurate all-atom model based on the well-known CHARMM force field, we obtain computationally the magnitudes of thermal vibrations of nearly 1000 structures. Our key findings are: (i) the magnitude of thermal vibrations, surprisingly, is nearly protein-independent, as a corollary to the universality for the vibrational spectra of globular proteins established earlier; (ii) the magnitude of thermal vibrations is small, less than 0.1 Å2 at 100 K; (iii) the percentage of thermal vibrations in B-factors is the lowest at low resolution and low temperature (<10%) but increases to as high as 60% for structures determined at high resolution and at room temperature. The significance of this work is that it provides for the first time, using an extremely large dataset, a thorough analysis of B-factors and their thermal and static disorder components. The results clearly demonstrate that structures determined at high resolution and at room temperature have the richest dynamics information. Since such structures are relatively rare in the PDB database, the work naturally calls for more such structures to be determined experimentally.

How anisotropic and isotropic atomic displacement parameters monitor protein covalent bonds rigidity: isotropic B-factors underestimate bond rigidity.

Under the assumption that covalent bonds are rigid, it is possible to compare the estimations of rigidity based on anisotropic and isotropic B-factors. This is done by computing the difference of the mean-square displacements (Delta-u) of atoms A and Z along the covalent bond A-Z, which must be close to zero for a rigid bond. The analysis of a high-quality set of protein structures, refined at a resolution better than (or equal to) 0.8 Angstroms, showed that Delta-u is significantly close to zero when anisotropic B-factors are used, with an average 60% Delta-u reduction. This reduction is larger for larger B-factors and this suggests that care should be taken in data-mining procedures that involve isotropic B-factors, especially at lower resolution, when anisotropic B-factors cannot be determined and when the average B-factor increases.

Re-examining the spectra of macromolecules. Current practice of spectral quasi B-factor flattening.

The analysis of structure factors in 3D cryo-EM Coulomb potential maps and their "enhancement" at the end of the reconstruction process is a well-established practice, normally referred to as sharpening. The aim is to increase contrast and, in this way, to help tracing the atomic model. The most common way to accomplish this enhancement is by means of the so-called B-factor correction, which applies a global filter to boost high frequencies with some dampening considerations related to noise amplification. The results are maps with a better visual aspect and a quasiflat spectrum at medium and high frequencies. This practice is so widespread that most map depositions in the Electron Microscopy Data Base (EMDB) only contain sharpened maps. Here, the use in cryoEM of global B-factor corrections is theoretically and experimentally analyzed. Results clearly illustrate that protein spectra present a falloff. Thus, spectral quasi-flattening may produce protein spectra with distortions when compared with experimental ones, this fact, combined with the practice of reporting only sharpened maps, generates a sub-optimal situation in terms of data preservation, reuse and reproducibility. Now that the field is more advanced, we put forward two suggestions: (1) to use methods which keep more faithfully the original experimental signal properties of macromolecules when "enhancing" the map, and (2) to further stress the need to deposit the original experimental maps without any postprocessing or sharpening, not only the enhanced maps. In the absence of access to these original maps data is lost, preventing their future analysis with new methods.

Structural compliance: A new metric for protein flexibility.

Proteins are the active players in performing essential molecular activities throughout biology, and their dynamics has been broadly demonstrated to relate to their mechanisms. The intrinsic fluctuations have often been used to represent their dynamics and then compared to the experimental B-factors. However, proteins do not move in a vacuum and their motions are modulated by solvent that can impose forces on the structure. In this paper, we introduce a new structural concept, which has been called the structural compliance, for the evaluation of the global and local deformability of the protein structure in response to intramolecular and solvent forces. Based on the application of pairwise pulling forces to a protein elastic network, this structural quantity has been computed and sometimes is even found to yield an improved correlation with the experimental B-factors, meaning that it may serve as a better metric for protein flexibility. The inverse of structural compliance, namely the structural stiffness, has also been defined, which shows a clear anticorrelation with the experimental data. Although the present applications are made to proteins, this approach can also be applied to other biomolecular structures such as RNA. This present study considers only elastic network models, but the approach could be applied further to conventional atomic molecular dynamics. Compliance is found to have a slightly better agreement with the experimental B-factors, perhaps reflecting its bias toward the effects of local perturbations, in contrast to mean square fluctuations. The code for calculating protein compliance and stiffness is freely accessible at https://jerniganlab.github.io/Software/PACKMAN/Tutorials/compliance.

Mobility of water and of protein atoms at the protein-water interface, monitored by anisotropic atomic displacement parameters, are largely uncorrelated.

A non-redundant set of 231 protein crystal structures refined at a resolution better than (or equal to) 1 Å was extracted from the Protein Data Bank and the degree of conformational rigidity at the protein-water interface was examined by means of the Hirshfeld test and by comparing the orientations of the anisotropic Us for contacting protein and water atoms. Contacts between protein and water atoms are more rigid that contacts between water atoms and the degree of rigidity increases for shorter contacts and for more hydrogen-bonded atoms. Nevertheless, water and protein atoms are not rigidly held together. On the contrary, they seem to have little influence on their mobility to such an extent that hydration water, different from the protein atoms, cannot be considered to be properly in the solid state.

Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser.

Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s-1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s-1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s-1. The µs-ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

Oversecretion and Overexpression of Nicotinamide Phosphoribosyltransferase/Pre-B Colony-Enhancing Factor/Visfatin in Inflammatory Bowel Disease Reflects the Disease Activity, Severity of Inflammatory Response and Hypoxia.

Nicotinamide phosphoribosyltransferase's (Nampt) association with inflammatory bowel disease (IBD) is unclear. The study was aimed at unraveling Nampt's clinical and diagnostic relevance. The serum concentration (Luminex-xMAP® technology) was measured in 113 patients with Crohn's disease (CD), 127 with ulcerative colitis (UC) and 60 non-IBD controls: 40 healthy individuals and 20 with irritable bowel syndrome (IBS). The leukocyte (44 CD/37 UC/19 IBS) and bowel expression (186 samples) was also evaluated (RT-qPCR). All were referred to IBD phenotype, activity, treatment, and inflammatory/nutritional/angiogenic/hypoxia indices. Serum-Nampt and leukocyte-Nampt were positively correlated and were more elevated in active-IBD than in IBS, with leukocyte-Nampt being a fair differential marker. Serum-Nampt in UC positively correlated with its clinical and endoscopic activity as well as with pro-inflammatory cytokines. Serum-Nampt ≤1.54 ng/mL was a good indicator of mucosal healing. The expression of Nampt was up-regulated both in inflamed and quiescent colon and reflected, similarly to leukocyte-Nampt, the clinical activity of IBD. Bowel-Nampt was independently associated with IL1B and hypoxia-inducible factor 1α (HIF1A) expression in inflamed bowel but with FGF2 expression in quiescent bowel. In summary, Nampt's elevation in IBD at local and systemic levels, and protein and mRNA levels, reflects IBD activity and is associated with inflammation, hypoxia (active) and tissue repair (inactive disease).

How large B-factors can be in protein crystal structures.

BACKGROUND: Protein crystal structures are potentially over-interpreted since they are routinely refined without any restraint on the upper limit of atomic B-factors. Consequently, some of their atoms, undetected in the electron density maps, are allowed to reach extremely large B-factors, even above 100 square Angstroms, and their final positions are purely speculative and not based on any experimental evidence. RESULTS: A strategy to define B-factors upper limits is described here, based on the analysis of protein crystal structures deposited in the Protein Data Bank prior 2008, when the tendency to allow B-factor to arbitrary inflate was limited. This B-factor upper limit (B_max) is determined by extrapolating the relationship between crystal structure average B-factor and percentage of crystal volume occupied by solvent (pcVol) to pcVol =100%, when, ab absurdo, the crystal contains only liquid solvent, the structure of which is, by definition, undetectable in electron density maps. CONCLUSIONS: It is thus possible to highlight structures with average B-factors larger than B_max, which should be considered with caution by the users of the information deposited in the Protein Data Bank, in order to avoid scientifically deleterious over-interpretations.

B-factor profile prediction for RNA flexibility using support vector machines.

Determining the flexibility of structured biomolecules is important for understanding their biological functions. One quantitative measurement of flexibility is the atomic Debye-Waller factor or temperature B-factor. Most existing studies are limited to temperature B-factors of proteins and their prediction. Only one method attempted to predict temperature B-factors of ribosomal RNA. Here, we developed and compared machine-learning techniques in prediction of temperature B-factors of RNAs. The best model based on Support Vector Machines yields Pearson's correction coefficient at 0.51 for fivefold cross validation and 0.50 for the independent test. Analysis of the performance indicates that the model has the best performance on rRNAs, tRNAs, and protein-bound RNAs, for long chains in particular. The server is available at http://sparks-lab.org/server/RNAflex. © 2017 Wiley Periodicals, Inc.

In silico prediction of novel residues involved in amyloid primary nucleation of human I56T and D67H lysozyme.

BACKGROUND: Amyloidogenic proteins are most often associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, but there are more than two dozen human proteins known to form amyloid fibrils associated with disease. Lysozyme is an antimicrobial protein that is used as a general model to study amyloid fibril formation. Studies aimed at elucidating the process of amyloid formation of lysozyme tend to focus on partial unfolding of the native state due to the relative instability of mutant amyloidogenic variants. While this is well supported, the data presented here suggest the native structure of the variants may also play a role in primary nucleation. RESULTS: Three-dimensional structural analysis identified lysozyme residues 21, 62, 104, and 122 as displaced in both amyloidogenic variants compared to wild type lysozyme. Residue interaction network (RIN) analysis found greater clustering of residues 112-117 in amyloidogenic variants of lysozyme compared to wild type. An analysis of the most energetically favored predicted dimers and trimers provided further evidence for a role for residues 21, 62, 104, 122, and 112-117 in amyloid formation. CONCLUSIONS: This study used lysozyme as a model to demonstrate the utility of combining 3D structural analysis with RIN analysis for studying the general process of amyloidogenesis. Results indicated that binding of two or more amyloidogenic lysozyme mutants may be involved in amyloid nucleation by placing key residues (21, 62, 104, 122, and 112-117) in proximity before partial unfolding occurs. Identifying residues in the native state that may be involved in amyloid formation could provide novel drug targets to prevent a range of amyloidoses.

Atomic displacement parameters in structural biology.

Atomic displacement parameters (ADPs, also known as B-factors), which depend on structural heterogeneity, provide a wide spectrum of information on protein structure and dynamics and find several applications, from protein conformational disorder prediction to protein thermostabilization, and from protein folding kinetics prediction to protein binding sites prediction. A crucial aspect is the standardization of the ADPs when comparisons between two or more protein crystal structures are made, since ADPs are differently affected by several factors, from crystallographic resolution to refinement protocols. A potential limitation to ADP analysis is the modern tendency to let ADPs to inflate up to extremely large values that have little physico-chemical meaning.

Engineering thermostable (R)-selective amine transaminase from Aspergillus terreus through in silico design employing B-factor and folding free energy calculations.

Amine transaminases have recently gained a lot of attention for the synthesis of chiral amines. Using (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) as a transaminase model, in silico design was applied employing B-factor and folding free energy (ΔΔGfold) calculations. Mutation sites were selected by targeting flexible regions with the greatest B-factors, and were substituted with amino acids that were determined by folding free energy calculations (ΔΔGfold < 0) to be more rigid than the original ones. By site-directed mutagenesis, we obtained four stabilized mutants (T130M, T130F, E133F and D134L) with improved stability from 19 candidates. Compared to the wild type, the best single mutant (T130M) showed an increase in thermal stability with a nearly 2.2-fold improvement of half-life (t1/2) at 40 °C and a 3.5 °C higher T1/210 min. The optimum catalytic temperature of T130F was increased by 10 °C. In addition, the T130M/E133F double mutant displayed the largest shift in thermostability with 3.3-fold improvement of t1/2 at 40 °C and a 5.0 °C higher T1/210 min. Modeling analysis showed that new hydrophobic interactions and hydrogen bonds might contribute to the observed thermostability improvement.