Conversion of cysteinyl residues to unnatural amino acid analogs. Examination in a model system

Improved and efficient techniques have led to an explosive growth in the application of site-directed mutagenesis to the study of enzymes. However, the limited availability of only those 20 amino acids that are translated by the genetic code has prevented the systematic variation of an amino acid's properties in order to define more precisely its role in the catalytic mechanism of an enzyme. An approach is being examined that combines the high specificity of site-directed mutagenesis with the flexibility of chemical modification to overcome these limitations. A set of reagents has been synthesized and reacted with a cysteine model to produce a series of amino acid structural analogs at appreciable rates and in good overall yields. The selective incorporation of these analogs in place of important functional amino acids in a protein will allow a more detailed examination of the role of that amino acid.     

A polyphosphate-lon protease complex in the adaptation of Escherichia coli to amino acid starvation

Cells must balance energy-efficient growth with the ability to adapt rapidly to sudden changes in their environment. For example, in an environment rich in amino acids, cells do not expend energy for making amino acid biosynthetic enzymes. However, if the environment becomes depleted of amino acids (nutritional downshift), cells will be exposed to a lack of both the amino acid biosynthetic enzymes and the amino acids required to make these enzymes. To solve this dilemma, cells must use their own proteins as sources of amino acids in response to the nutritional downshift. Once amino acid biosynthetic enzymes start to accumulate, the cell is able to produce its own amino acids, and a new growth phase begins. In Escherichia coli, amino acid starvation leads to the accumulation of an unusual molecule, polyphosphate (polyP), a linear polymer of many hundreds of orthophosphate residues. Protein degradation in this bacterium appears to be triggered by the accumulation of polyP. PolyP forms a complex with the ATP-dependent Lon protease. The formation of a complex then enables Lon to degrade free ribosomal proteins. Certain very abundant ribosomal proteins can be the sacrificial substrates targeted for degradation at the onset of the downshift. Here I propose to call the polyP-Lon complex the "stringent protease," and I discuss new insights of protein degradation control in bacteria.     

Efficient prediction of nucleic acid binding function from low-resolution protein structures

Structural genomics projects as well as ab initio protein structure prediction methods provide structures of proteins with no sequence or fold similarity to proteins with known functions. These are often low-resolution structures that may only include the positions of C alpha atoms. We present a fast and efficient method to predict DNA-binding proteins from just the amino acid sequences and low-resolution, C alpha-only protein models. The method uses the relative proportions of certain amino acids in the protein sequence, the asymmetry of the spatial distribution of certain other amino acids as well as the dipole moment of the molecule. These quantities are used in a linear formula, with coefficients derived from logistic regression performed on a training set, and DNA-binding is predicted based on whether the result is above a certain threshold. We show that the method is insensitive to errors in the atomic coordinates and provides correct predictions even on inaccurate protein models. We demonstrate that the method is capable of predicting proteins with novel binding site motifs and structures solved in an unbound state. The accuracy of our method is close to another, published method that uses all-atom structures, time-consuming calculations and information on conserved residues.     

Food protein fragments are regulatory oligopeptides

Until recently food proteins were considered to be an energy source and a source of essential and nonessential amino acids required for protein synthesis and precursors of many vital biomolecules. However, we assumed earlier that food protein fragments might perform some regulatory functions. The theoretical justification for this assumption is advanced in this work. In the present work, the primary structures of protein fragments were compared with amino acid sequences of known natural regulatory oligopeptides in silico. It is shown that fragments formed as a result of animal food protein cleavage by proteolytic enzymes can exist in the gastrointestinal tract for a long time. Many of them are enzyme inhibitors, regulators of nervous, endocrine, and immune system, and possess antimicrobial and other activities. It has also been shown that the lifetime of fragments before their cleavage in the gastrointestinal tract could be enough for performing corrective functions. Thus, as a result of food protein fragmentation a dynamic pool of exogenous regulatory oligopeptides with functions changing as shorter fragments are generated may form. The detection of an endogenous-exogenous pool of regulatory molecules expands the significance and content of the Ashmarin-Obukhova hypothesis on a functional continuum of natural oligopeptides. The possible practical importance of these results is noted.     

Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes

Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Vorono? tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions.     

Predicting Protein Function and Binding Profile via Matching of Local Evolutionary and Geometric Surface Patterns

Inferring protein functions from structures is a challenging task, as a large number of orphan protein structures from structural genomics project are now solved without their biochemical functions characterized. For proteins binding to similar substrates or ligands and carrying out similar functions, their binding surfaces are under similar physicochemical constraints, and hence the sets of allowed and forbidden residue substitutions are similar. However, it is difficult to isolate such selection pressure due to protein function from selection pressure due to protein folding, and evolutionary relationship reflected by global sequence and structure similarities between proteins is often unreliable for inferring protein function. We have developed a method, called pevoSOAR (pocket-based evolutionary search of amino acid residues), for predicting protein functions by solving the problem of uncovering amino acids residue substitution pattern due to protein function and separating it from amino acids substitution pattern due to protein folding. We incorporate evolutionary information specific to an individual binding region and match local surfaces on a large scale with millions of precomputed protein surfaces to identify those with similar functions. Our pevoSOAR method also generates a probablistic model called the computed binding a profile that characterizes protein-binding activities that may involve multiple substrates or ligands. We show that our method can be used to predict enzyme functions with accuracy. Our method can also assess enzyme binding specificity and promiscuity. In an objective large-scale test of 100 enzyme families with thousands of structures, our predictions are found to be sensitive and specific: At the stringent specificity level of 99.98%, we can correctly predict enzyme functions for 80.55% of the proteins. The overall area under the receiver operating characteristic curve measuring the performance of our prediction is 0.955, close to the perfect value of 1.00. The best Matthews coefficient is 86.6%. Our method also works well in predicting the biochemical functions of orphan proteins from structural genomics projects.     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

Discarding functional residues from the substitution table improves predictions of active sites within three-dimensional structures

Substitutions of individual amino acids in proteins may be under very different evolutionary restraints depending on their structural and functional roles. The Environment Specific Substitution Table (ESST) describes the pattern of substitutions in terms of amino acid location within elements of secondary structure, solvent accessibility, and the existence of hydrogen bonds between side chains and neighbouring amino acid residues. Clearly amino acids that have very different local environments in their functional state compared to those in the protein analysed will give rise to inconsistencies in the calculation of amino acid substitution tables. Here, we describe how the calculation of ESSTs can be improved by discarding the functional residues from the calculation of substitution tables. Four categories of functions are examined in this study: protein–protein interactions, protein–nucleic acid interactions, protein–ligand interactions, and catalytic activity of enzymes. Their contributions to residue conservation are measured and investigated. We test our new ESSTs using the program CRESCENDO, designed to predict functional residues by exploiting knowledge of amino acid substitutions, and compare the benchmark results with proteins whose functions have been defined experimentally. The new methodology increases the Z-score by 98% at the active site residues and finds 16% more active sites compared with the old ESST. We also find that discarding amino acids responsible for protein–protein interactions helps in the prediction of those residues although they are not as conserved as the residues of active sites. Our methodology can make the substitution tables better reflect and describe the substitution patterns of amino acids that are under structural restraints only.     

Direct protein–protein conjugation by genetically introducing bioorthogonal functional groups into proteins

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein–protein conjugates has gained research interest. In this study, site-specific protein–protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein–protein conjugates and protein complexes of biochemical significance.     

Conformational preference functions for predicting helices in membrane proteins

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the α-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in α-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 α-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known α-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements. © 1993 John Wiley & Sons, Inc.     

New enzymes for old: redesigning the coenzyme and substrate specificities of glutathione reductase.

A set of amino acid side chains that confer specificity for the coenzyme NADPH and the substrate glutathione in the flavoprotein disulphide oxidoreductase, glutathione reductase, has been identified. Systematic replacement of these amino acid residues in the coenzyme-binding site switches the specificity of the enzyme from its natural strong preference for NADPH to a marked preference for NADH. The amino acids replaced all lie in a structural motif within the dinucleotide-binding domain of the protein. Since this domain is a feature common to most dehydrogenases (reductases) that use nicotinamide coenzymes, it may be that the coenzyme specificities of all such enzymes can be manipulated in this way. Similarly, amino acid residues involved in the selective recognition of trypanothione by trypanothione reductase, an enzyme related to glutathione reductase and exclusive to trypanosomatids, were identified. Suitable mutation of the corresponding residues in E. coli glutathione reductase switched its substrate specificity towards trypanothione. A better understanding of the substrate specificity of these enzymes could open up a route to the chemotherapy of trypanosomal infections.     

Local sequence‐structure relationships in proteins

We seek to understand the interplay between amino acid sequence and local structure in proteins. Are some amino acids unique in their ability to fit harmoniously into certain local structures? What is the role of sequence in sculpting the putative native state folds from myriad possible conformations? In order to address these questions, we represent the local structure of each C_α atom of a protein by just two angles, θ and μ, and we analyze a set of more than 4,000 protein structures from the PDB. We use a hierarchical clustering scheme to divide the 20 amino acids into six distinct groups based on their similarity to each other in fitting local structural space. We present the results of a detailed analysis of patterns of amino acid specificity in adopting local structural conformations and show that the sequence‐structure correlation is not very strong compared with a random assignment of sequence to structure. Yet, our analysis may be useful to determine an effective scoring rubric for quantifying the match of an amino acid to its putative local structure.     

Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

Computational exploration of the network of sequence flow between protein structures

We investigate small sequence adjustments (of one or a few amino acids) that induce large conformational transitions between distinct and stable folds of proteins. Such transitions are intriguing from evolutionary and protein‐design perspectives. They make it possible to search for ancient protein structures or to design protein switches that flip between folds and functions. A network of sequence flow between protein folds is computed for representative structures of the Protein Data Bank. The computed network is dense, on an average each structure is connected to tens of other folds. Proteins that attract sequences from a higher than expected number of neighboring folds are more likely to be enzymes and alpha/beta fold. The large number of connections between folds may reflect the need of enzymes to adjust their structures for alternative substrates. The network of the Cro family is discussed, and we speculate that capacity is an important factor (but not the only one) that determines protein evolution. The experimentally observed flip from all alpha to alpha + beta fold is examined by the network tools. A kinetic model for the transition of sequences between the folds (with only protein stability in mind) is proposed. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Exploring amino acid functions in a deep mutational landscape

Amino acids fulfil a diverse range of roles in proteins, each utilising its chemical properties in different ways in different contexts to create required functions. For example, cysteines form disulphide or hydrogen bonds in different circumstances and charged amino acids do not always make use of their charge. The repertoire of amino acid functions and the frequency at which they occur in proteins remains understudied. Measuring large numbers of mutational consequences, which can elucidate the role an amino acid plays, was prohibitively time‐consuming until recent developments in deep mutational scanning. In this study, we gathered data from 28 deep mutational scanning studies, covering 6,291 positions in 30 proteins, and used the consequences of mutation at each position to define a mutational landscape. We demonstrated rich relationships between this landscape and biophysical or evolutionary properties. Finally, we identified 100 functional amino acid subtypes with a data‐driven clustering analysis and studied their features, including their frequencies and chemical properties such as tolerating polarity, hydrophobicity or being intolerant of charge or specific amino acids. The mutational landscape and amino acid subtypes provide a foundational catalogue of amino acid functional diversity, which will be refined as the number of studied protein positions increases.     

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.     

The 4-oxalocrotonate tautomerase family of enzymes: how nature makes new enzymes using a beta-alpha-beta structural motif

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers. The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy. Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base. In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies. Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink. Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50. 4-OT is also the title enzyme of the 4-OT family of enzymes. The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif. The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position. Characterization of representative members has uncovered mechanistic and structural diversity. A new activity, a trans-3-chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities. Two new structures have also been found, along with the 4-OT hexamer. The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer. Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity. Amplification of this activity could have produced the full-fledged dehalogenase. The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes.     

Dietary supplements of mixtures of indispensable amino acids lacking threonine, phenylalanine or histidine increase the activity of hepatic threonine dehydrogenase, phenylalanine hydroxylase or histidase, respectively, and prevent growth depressions in chicks caused by dietary excesses of threonine, phenylalanine, or histidine*

Experiments were carried out to determine whether the addition of a mixture of indispensable amino acids (IAA) lacking in threonine, phenylalanine or histidine, respectively, to a nutritionally complete diet would increase the hepatic activities of the rate-limiting enzymes for catabolism of threonine, phenylalanine or histidine and prevent the adverse effects of the amino acid on growth when the dietary level of the amino acid is excessive. Week old Leghorn chicks were fed semi-purified diets containing 19% crude protein to which were added no IAA supplement or 10% crude protein from an IAA mix and 5 graded levels of either L-threonine, L-phenylalanine or L-histidine in a 2 x 5 factorial arrangement of treatments. Each amino acid was investigated in a separate experiment involving four replicate pens (seven chicks each) per diet. Weight gains and feed consumptions were determined on the fourteenth day of each experiment. The groups receiving no excess, and 1.0% or 2.0% excesses of amino acids were sampled on the fifteenth day for enzyme activities and plasma amino acid concentrations. Weight gain and/or feed consumption were lower, and plasma concentrations of threonine, phenylalanine and histidine were higher, in chicks receiving 1.5 to 2.0% dietary additions of threonine, phenylalanine, and histidine, respectively, than in chicks that did not receive these amino acids. Chicks that received the amino acids in diets that also contained the IAA supplement had better growth and feed consumption, lower plasma concentrations of threonine, phenylalanine or histidine, higher plasma concentrations of other indispensable amino acids, and higher activities of threonine dehydrogenase, phenylalanine hydroxylase, and histidase than chicks receiving excess amino acids in the absence of IAA supplements. We conclude that the dietary level of protein, not the dietary level of individual amino acids, is the primary determinant of the activity of amino acid degrading enzymes in liver. The increased activity of these enzymes may be the mechanism by which dietary protein alleviates the adverse effects of excessive levels of individual amino acids.     

Extension of biochemical functions by the introduction of nonnatural amino acids and artificial nucleic acid analogs.

Extension of biochemical functions has been attempted by introducing nonatural amino acids and artificial nucleic acid analogs. Nonnatural amino acids have been linked to tRNAs and the amino-acylated tRNAs were added to E. coli in vitro protein synthesizing system to produce nonnatural mutant proteins. The positions of the nonnatural amino acids have been assigned by the 4-base codons, like CGGG and AGGU. The extended codons have been introduced at a specific position or at random positions on a DNA. In the latter case, a DNA library that contains a single 4-base codon at random positions can be obtained. The combination of these new techniques opens a way to the introduction of artificial functions to biochemical systems.     

Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein.