Identifying metal binding amino acids based on backbone geometries as a tool for metalloprotein engineering

Metal cofactors within proteins perform a versatile set of essential cellular functions. In order to take advantage of the diverse functionality of metalloproteins, researchers have been working to design or modify metal binding sites in proteins to rationally tune the function or activity of the metal cofactor. This study has performed an analysis on the backbone atom geometries of metal‐binding amino acids among 10 different metal binding sites within the entire protein data bank. A set of 13 geometric parameters (features) was identified that is capable of predicting the presence of a metal cofactor in the protein structure with overall accuracies of up to 97% given only the relative positions of their backbone atoms. The decision tree machine‐learning algorithm used can quickly analyze an entire protein structure for the presence of sets of primary metal coordination spheres upon mutagenesis, independent of their original amino acid identities. The methodology was designed for application in the field of metalloprotein engineering. A cluster analysis using the data set was also performed and demonstrated that the features chosen are useful for identifying clusters of structurally similar metal‐binding sites.     

Predicting mutant outcome by combining deep mutational scanning and machine learning

Deep mutational scanning provides unprecedented wealth of quantitative data regarding the functional outcome of mutations in proteins. A single experiment may measure properties (eg, structural stability) of numerous protein variants. Leveraging the experimental data to gain insights about unexplored regions of the mutational landscape is a major computational challenge. Such insights may facilitate further experimental work and accelerate the development of novel protein variants with beneficial therapeutic or industrially relevant properties. Here we present a novel, machine learning approach for the prediction of functional mutation outcome in the context of deep mutational screens. Using sequence (one-hot) features of variants with known properties, as well as structural features derived from models thereof, we train predictive statistical models to estimate the unknown properties of other variants. The utility of the new computational scheme is demonstrated using five sets of mutational scanning data, denoted “targets”: (a) protease specificity of APPI (amyloid precursor protein inhibitor) variants; (b-d) three stability related properties of IGBPG (immunoglobulin G-binding β1 domain of streptococcal protein G) variants; and (e) fluorescence of GFP (green fluorescent protein) variants. Performance is measured by the overall correlation of the predicted and observed properties, and enrichment—the ability to predict the most potent variants and presumably guide further experiments. Despite the diversity of the targets the statistical models can generalize variant examples thereof and predict the properties of test variants with both single and multiple mutations.     

Characterization of hen egg white- and yolk-riboflavin binding proteins and amino acid sequence of egg white-riboflavin binding protein

White- and Yolk-riboflavin binding proteins were isolated from hen eggs, and characterized as to their chemical properties. White- and Yolk-RBPs had almost same amino acid compositions except for glutamic acid, but their carbohydrate compositions were different from each other. The complete amino acid sequence of White-RBP was determined by conventional methods. White-RBP comprised 219 amino acid residues, and the amino-terminus was pyroglutamic acid (pyrrolidonecarboxylic acid). Two amino acids, lysine and asparagine, were found at the fourteenth residue from the amino-terminus. Carbohydrate chains were linked to asparagine residues at positions 36 and 147. Both White- and Yolk-RBPs were phosphorylated. In White-RBP either six or seven of nine serine residues between Ser(185) and Ser(197) were phosphorylated. The amino acid sequences around phosphoserines showed that phosphorylation might occur at a serine residue in one of the following sequences; Ser-X-Glu or Ser-X-Ser(P).     

Functional aspects of co-variant surface charges in an antibody fragment

A mutational analysis of three co-variant pairs of residues, located at the surface of a single-chain fragment, variable (scFv), remote from the antigen-binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen-binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium-chloride denaturation was similar. These observations indicate that the mutations did not affect antigen-binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half-life in periplasmic extracts (<4 h to >16 h at 25 degrees C). The deleterious effect on half-life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co-variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half-life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.     

The order of PDZ3 and TrpCage in fusion chimeras determines their properties—a biophysical characterization

Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structural characteristics of individual domains in a new context of artificial fusion proteins may help to reveal the rules of internal and external domain communication. Moreover, this could also help explain the mechanism of such communication and address how the mutual allosteric effect plays a role in a such multi‐domain protein system. The simple model system of the two‐domain fusion protein investigated in this work consisted of a well‐folded PDZ3 domain and an artificially designed small protein domain called Tryptophan Cage (TrpCage). Two fusion proteins with swapped domain order were designed to study their structural and functional features as well as their biophysical properties. The proteins composed of PDZ3 and TrpCage, both identical in amino acid sequence but different in composition (PDZ3‐TrpCage, TrpCage‐PDZ3), were studied using circualr dichroism (CD) spectrometry, analytical ultracentrifugation, and molecular dynamic simulations. The biophysical analysis uncovered different structural and denaturation properties of both studied proteins, revealing their different unfolding pathways and dynamics.     

The cellular modifier MOAG‐4/SERF drives amyloid formation through charge complementation

While aggregation‐prone proteins are known to accelerate aging and cause age‐related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG‐4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid‐promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG‐4 to neutralize charge. Our data indicate that MOAG‐4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation‐prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age‐related protein toxicity.     

The influence of site-specificity of single amino acid substitutions on electrophoretic separation of yeast iso-1-cytochromec

Summary This study dealt with the ability of non-denaturing gel electrophoresis to separate iso-1-cytochromec with single amino acid replacements isolated from revertants of variouscyc1 nonsense mutants of the yeastSaccharomyces cerevisiae. A total of 28 different iso-1-cytochromesc with single amino acid substitutions of one of seven amino acids at six positions were examined on nondenaturing polyacrylamide gels at pH 4.8. Each of these iso-1-cytochromesc exhibited 1 of 16 distinct electrophoretic mobilities. We could distinguish the majority of iso-1-cytochromesc, even those having the same replacement at different sites and those having different replacements that resulted in the same net charge. These results provide confirmation of the importance of site-specific effects on the electrophoretic mobility, and presumably other properties, of proteins differing in sequence by as little as one amino acid. They demonstrate that nondenaturing electrophoresis is able to separate the majority of, but not all, proteins differing by single amino acids.     

Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues

As Fran?ois Jacob pointed out over 30 years ago, evolution is a tinkering process, and, as such, relies on the genetic diversity produced by mutation subsequently shaped by Darwinian selection. However, there is one implicit assumption that is made when studying this tinkering process; it is typically assumed that all amino acid residues are equally likely to mutate or to result from a mutation. Here, by reconstructing ancestral sequences and computing mutational probabilities for all the amino acid residues, we refute this assumption and show extensive inequalities between different residues in terms of their mutational activity. Moreover, we highlight the importance of the genetic code and physico-chemical properties of the amino acid residues as likely causes of these inequalities and uncover serine as a mutational hot spot. Finally, we explore the consequences that these different mutational properties have on phosphorylation site evolution, showing that a higher degree of evolvability exists for phosphorylated threonine and, to a lesser extent, serine in comparison with tyrosine residues. As exemplified by the suppression of serine's mutational activity in phosphorylation sites, our results suggest that the cell can fine-tune the mutational activities of amino acid residues when they reside in functional protein regions.     

Percent of highly immunogenic amino acid residues forming B-cell epitopes is higher in homologous proteins encoded by GC-rich genes

We analyzed the dependence of the percent of highly immunogenic amino acid residues included in B-cell epitopes of homologous proteins on the GC-content (G+C) of genes coding for them in twenty-seven lineages of proteins (and subsequent genes), which belong to seven Varicello and five Simplex viruses. We found out that proteins encoded by genes of a high GC-content usually contain more targets for humoral immune response than their homologs encoded by GC-poor genes. This tendency is characteristic not only to the lineages of glycoproteins, which are the main targets for humoral immune response against Simplex and Varicello viruses, but also to the lineages of capsid proteins and even "housekeeping" enzymes. The percent of amino acids included in linear B-cell epitopes has been predicted for 324 proteins by BepiPred algorithm (www.cbs.dtu.dk/services/BepiPred), the percent of highly immunogenic amino acids included in discontinuous B-cell epitopes and the percent of exposed amino acid residues have been predicted by Epitopia algorithm (http://epitopia.tau.ac.il/). Immunological consequences of the directional mutational GC-pressure are mostly due to the decrease in the total usage of highly hydrophobic amino acids and due to the increase in proline and glycine levels of usage in proteins. The weaker the negative selection on amino acid substitutions caused by symmetric mutational pressure, the higher the slope of direct dependence of the percent of highly immunogenic amino acids included in B-cell epitopes on G+C.     

The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins

beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse beta-lactoglobulin II is unique in that it contains 166 amino acids. The additional four amino acids represent an insertion between positions 116 and 117 of other beta-lactoglobulins so far sequenced, including horse beta-lactoglobulin I. Sequence comparison of beta-lactoglobulins I and II from horse colostrum reveals 48 amino acid substitutions (30%). Such a diversity between members of the beta-lactoglobulin gene family has not been encountered before. Sequence comparison with bovine beta-lactoglobulin A shows 85 amino acid replacements accounting for 53% of the residues. The structural homology with human retinol-binding protein may reveal similar biological functions and clues to the origin of milk proteins.     

High diversity in Delta variant across countries revealed by genome‐wide analysis of SARS‐CoV‐2 beyond the Spike protein

The highly contagious Delta variant of SARS‐CoV‐2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant’s Spike protein, the mutational landscape of the rest of the SARS‐CoV‐2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS‐CoV‐2 proteins from nearly 2 million SARS‐CoV‐2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle‐associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant’s proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.     

A useful epitope tag derived from maltose binding protein

Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross‐reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co‐crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S‐transferase protein were engineered in order to identify the smallest fragment still recognized by the α‐MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino‐acid tag.     

Assessment of the optimization of affinity and specificity at protein–DNA interfaces

The biological functions of DNA-binding proteins often require that they interact with their targets with high affinity and/or high specificity. Here, we describe a computational method that estimates the extent of optimization for affinity and specificity of amino acids at a protein–DNA interface based on the crystal structure of the complex, by modeling the changes in binding-free energy associated with all individual amino acid and base substitutions at the interface. The extent to which residues are predicted to be optimal for specificity versus affinity varies within a given protein–DNA interface and between different complexes, and in many cases recapitulates previous experimental observations. The approach provides a complement to traditional methods of mutational analysis, and should be useful for rapidly formulating hypotheses about the roles of amino acid residues in protein–DNA interfaces.     

Application of genetic semihomology algorithm to theoretical studies on various protein families.

Several protein families of different nature were studied for genetic relationship, correct alignment at non-homologous fragments, optimal sequence consensus construction, and confirmation of their actual relevance. A comparison of the genetic semihomology approach with statistical approaches indicates a high accuracy and cognition significance of the former. This is particularly pronounced in the study of related proteins that show a low degree of homology. The sequence multiple alignments were verified and corrected with respect to the questionable, non-homologous fragments. The verified alignments were the basis for consensus sequence formation. The frequency of six-codon amino acids occurrence versus position variability was studied and their possible role in amino acid mutational exchange at variable positions is discussed.     

Protein and cellular engineering with unnatural amino acids

Proteins are the central functional constituents in all living organisms ranging from viruses, bacteria, yeast, and plants to mammals. All of these biopolymers that are formed by natural biosynthetic pathways are composed of a genetically determined sequence of the 20 so-called natural amino acids. The physical and chemical properties of proteins are a reflection of the side chains of each of the component amino acids. However, for some purposes it would be very desireable to have amino acids with side chains of various selected physical chemical properties, such as a keto group, a crosslinker, or a NMR probe group, incorporated into the protein. Although chemical and biochemical methods for modifying amino acid moieties in proteins have been achieved, recent successes in incorporating unnatural amino acids in vivo open entirely new avenues for determining protein functions in vivo and for the creation of unnatural proteins with novel functionalities. Several examples by employing the novel activity of unnatural amino acids have shown significant roles in both basic research and biotechnology.     

Evolution of the amino acid substitution in the mammalian myoglobin gene

Summary Multivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.     

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).     

Second codon positions of genes and the secondary structures of proteins. Relationships and implications for the origin of the genetic code

The nucleotide frequencies in the second codon positions of genes are remarkably different for the coding regions that correspond to different secondary structures in the encoded proteins, namely, helix, beta-strand and aperiodic structures. Indeed, hydrophobic and hydrophilic amino acids are encoded by codons having U or A, respectively, in their second position. Moreover, the beta-strand structure is strongly hydrophobic, while aperiodic structures contain more hydrophilic amino acids. The relationship between nucleotide frequencies and protein secondary structures is associated not only with the physico-chemical properties of these structures but also with the organisation of the genetic code. In fact, this organisation seems to have evolved so as to preserve the secondary structures of proteins by preventing deleterious amino acid substitutions that could modify the physico-chemical properties required for an optimal structure.     

Facile Method for High-throughput Identification of Stabilizing Mutations

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. However, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 °C increase in thermal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.