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.     

The joint evolution of defence and inducibility against natural enemies

The aminoacyl-tRNA synthetases (aaRSs) ensure the fidelity of the translation of the genetic code, covalently attaching appropriate amino acids to the corresponding nucleic acid adaptor molecules-tRNA. The fundamental role of aminoacylation reaction catalysed by aaRSs implies that representatives of the family are thought to be among the earliest proteins to appear. Based on sequence analysis and catalytic domain structure, aaRSs have been partitioned into two classes of 10 enzymes each. However, based on the structural and sequence data only, it will not be easily understood that the present partitioning is not governed by chance. Our findings suggest that organization of amino acid biosynthetic pathways and clustering of aaRSs into different classes are intimately related to one another. A plausible explanation for such a relationship is dictated by early link between aaRSs and amino acids biosynthetic proteins. The aaRSs catalytic cores are highly relevant to the ancient metabolic reactions, namely, amino acids and cofactors biosynthesis. In particular we show that class II aaRSs mostly associated with the primordial amino acids, while class I aaRSs are usually related to amino acids evolved lately. Reasoning from this we propose a possible chronology of genetic code evolution.     

Amino acid chalcogen analogues as tools in peptide and protein research

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur‐containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl‐tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl‐ and phenylalanyl‐tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl‐tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.     

On How Many Fundamental Kinds of Cells are Present on Earth: Looking for Phylogenetic Traits that Would Allow the Identification of the Primary Lines of Descent

The phylogenetic analyses as far as the identification of the number of domains of life is concerned have not reached a clear conclusion. In the attempt to improve this circumstance, I introduce the concept that the amino acids codified in the genetic code might be of markers with outstanding phylogenetic power. In particular, I hypothesise the existence of a biosphere populated, for instance, by three groups of organisms having different genetic codes because codifying at least a different amino acid. Evidently, these amino acids would mark the proteins that are present in the three groups of organisms in an unambiguous way. Therefore, in essence, this mark would not be other than the one that we usually try to make in the phylogenetic analyses in which we transform the protein sequences in phylogenetic trees, for the purpose to identify, for example, the domains of life. Indeed, this mark would allow to classify proteins without performing phylogenetic analyses because proteins belonging to a group of organisms would be recognisable as marked in a natural way by at least a different amino acid among the diverse groups of organisms. This conceptualisation answers the question of how many fundamental kinds of cells have evolved from the Last Universal Common Ancestor (LUCA), as the genetic code has unique proprieties that make the codified amino acids excellent phylogenetic markers. The presence of the formyl-methionine only in proteins of bacteria would mark them and would identify these as domain of life. On the other hand, the presence of pyrrolysine in the genetic code of the euryarchaeota would identify them such as another fundamental kind of cell evolved from the LUCA. Overall, the phylogenetic distribution of formyl-methionine and pyrrolysine would identify at least two domains of life—Bacteria and Archaea—but their number might be actually four; that is to say, Bacteria, Euryarchaeota, archeobacteria that are not euryarchaeota and Eukarya. The usually accepted domains of life represented by Bacteria, Archaea and Eukarya are not compatible with the phylogenetic distribution of these two amino acids and therefore this last classification might be mistaken.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Protein psychrophilicity: role of residual structural properties in adaptation of proteins to low temperatures

In order to investigate the structural distribution responsible for protein psychrophilicity, a systematic comparative analysis of 13 pairs of psychrophilic and mesophilic proteins is reported. Three kinds of residue structural states such as exposed, intermediate and buried were considered for analyzing the structural patterns of single amino acids and amino acids in different groups. The statistical test revealed that higher frequency in exposed state of Ala, higher frequency in intermediate state of His, lower frequency in buried state of Lys, lower frequency in exposed state of Gln, higher frequency in exposed state and in intermediate state of Thr, higher frequency in exposed and intermediate state of tiny and small amino acids groups could be critical factors related with protein psychrophilicity. Such structure-based differences of residual properties would help to develop a strategy for designing psychrophilic proteins.     

A review of adsorption of amino acids on minerals: Was it important for origin of life?

Minerals more readily adsorb amino acids with charged R groups than uncharged R groups, so that the incorporation of amino acids with charged R groups into peptides would be more frequent than for amino acids with uncharged R groups. However, 74% of the amino acids in the proteins of modern organisms contain uncharged R groups. Thus, what could have been the mechanisms that produced peptides/proteins with more amino acids with uncharged R groups than precursors with charged R groups? Should we expect the composition of amino acids adsorbed on minerals to be similar to those of present proteins? Was the adsorption of amino acids on minerals important for the origin of life? The lipid world offers an alternative view of origin of life. Liposomes contributed to elongation of peptides as well as select hydrophobic amino acids and peptides. These experiments could be showing the mechanism, which hydrophobic amino acids have been selected. However, liposomes have no influence on the stereoselectivity in the oligomerization of amino acids. In the present paper, several other mechanisms are also discussed that could produce peptides with a greater proportion of amino acids with uncharged R groups.     

Expansion of the genetic code in yeast: making life more complex

Proteins account for the catalytic and structural versatility displayed by all cells, yet they are assembled from a set of only 20 common amino acids. With few exceptions, only 61 nucleotide triplets also direct incorporation of these amino acids. Endeavors to expand the genetic code recently progressed to nucleus-containing cells, after Chin et al.1 transferred Escherichia coli genes for a mutant tyrosine-adaptor molecule and its synthetase into Saccharomyces cerevisiae. Transformed yeast cells were produced that exhibit efficient site-specific incorporation of non-biotic amino acids into proteins. This makes it likely that code complexity can be elevated experimentally in mammals.     

A possible molecular metric for biological evolvability

Proteins manifest themselves as phenotypic traits, retained or lost in living systems via evolutionary pressures. Simply put, survival is essentially the ability of a living system to synthesize a functional protein that allows for a response to environmental perturbations (adaptation). Loss of functional proteins leads to extinction. Currently there are no universally applicable quantitative metrics at the molecular level for either measuring 'evolvability' of life or for assessing the conditions under which a living system would go extinct and why. In this work, we show emergence of the first such metric by utilizing the recently discovered stoichiometric margin of life for all known naturally occurring (and functional) proteins. The constraint of having well-defined stoichiometries of the 20 amino acids in naturally occurring protein sequences requires utilization of the full scope of degeneracy in the genetic code, i.e. usage of all codons coding for an amino acid, by only 11 of the 20 amino acids. This shows that the non-availability of individual codons for these 11 amino acids would disturb the fine stoichiometric balance resulting in non-functional proteins and hence extinction. Remarkably, these amino acids are found in close proximity of any given amino acid in the backbones of thousands of known crystal structures of folded proteins. On the other hand, stoichiometry of the remaining 9 amino acids, found to be farther/distal from any given amino acid in backbones of folded proteins, is maintained independent of the number of codons available to synthesize them, thereby providing some robustness and hence survivability.     

Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein.

When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the standard 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophysical probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are observed when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances associated with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addition, 19F and [1H-13C]-HMQC NMR experiments have been utilized with paramagnetic line broadening and K2PtCl4 reactivity experiments to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently determined crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.     

Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

Screening of functional proteins from a random‐sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random‐sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random‐sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random‐sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120‐amino acid, random‐sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random‐sequence proteins arbitrarily chosen from these libraries. We found that random‐sequence proteins constructed with the 12‐member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20‐member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids.     

Effect of low-complexity regions on protein structure determination

It has been previously shown that protein sequences containing a quasi-repetitive assortment of amino acids are common in genomes and databases such as Swiss-Prot but are under-represented in the structure-based Protein Data Bank (PDB). Structural genomics groups have been using the absence of these “low-complexity” sequences for several years as a way to select proteins that have a good chance of successful structure determination. In this study, we examine the data deposited in the PDB as well as the available data from structural genomics groups in TargetDB and PepcDB to reveal interesting trends that could be taken into consideration when using low-complexity sequences as part of the target selection process.     

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.     

Zinc coordination sphere in biochemical zinc sites

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.     

Correlations of amino acids in proteins

A correlation analysis among 20 amino acids is performed for four protein structural classes (, β, /β, and +β) in a total of 204 proteins. The correlation relationships among amino acids can be classified into the following four types: (1) strong positive correlation, (2) strong negative correlation, (3) weak correlation, and (4) no correlation. The correlation relationships are different for different proteins and are correlated with the features of their structural classes. The amino acids with the weak correlation relationship can be treated as the independent basis functions for the space where proteins are defined. The amino acids with large correlation coefficients are linear correlative with each other and they are not independent. The strong correlation among amino acids reflects their mutual constrained relationship, as exhibited by their relevant structural features. The information obtained through the correlation analysis is used for predicting protein structural classes and a better prediction quality is obtained than that by the simple geometry distance methods without taking into account the correlation effects.     

Combining hydrophobicity and helicity: a novel approach to membrane protein structure prediction

In spite of the overwhelming numbers and critical biological functions of membrane proteins, only a few have been characterized by high-resolution structural techniques. From the structures that are known, it is seen that their transmembrane (TM) segments tend to fold most often into alpha-helices. To evaluate systematically the features of these TM segments, we have taken two approaches: (1) using the experimentally-measured residence behavior of specifically designed hydrophobic peptides in RP-HPLC, a scale was derived based directly on the properties of individual amino acids incorporated into membrane-interactive helices: and (2) the relative alpha-helical propensity of each of the 20 amino acids was measured in the organic non-polar environment of n-butanol. By combining the resulting hydrophobicity and helical propensity data, in conjunction with consideration of the 'threshold hydrophobicity' required for spontaneous membrane integration of protein segments, an approach was developed for prediction of TM segments wherein each must fulfill the dual requirements of hydrophobicity and helicity. Evaluated against the available high-resolution structural data on membrane proteins, the present combining method is shown to provide accurate predictions for the locations of TM helices. In contrast, no segment in soluble proteins was predicted as a 'TM helix'.     

Structured proteins and proteins with internal disorder

The point of view that a uniquely folded protein tertiary structure is required for the protein functioning has been prevailing in the literature quite recently. However of lately it has been found that many proteins in a cell have no such structure in an isolated state, though they have a well-defined function in physiological conditions. These proteins were named as proteins with natural or internal disorder. The portion of disordered regions in such proteins may vary from a sequence of several amino acids to a completely disordered sequence containing from tens to hundreds of amino acids. The main difference of these proteins from the structured (globular) ones is that they have no unique tertiary structure in an isolated state and acquire it after interaction with their partners. Their conformation in such a complex depends on the interacting partner and not only on their own amino acid sequence, which is specific for structured (globular) proteins. The problem of structural and functional relations in the structured proteins and proteins with internal disorder is discussed in this review. The complexity of the problem and its potential solutions are illustrated by the example of elongation factors EFlA.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.