Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Computational analysis of sequence selection mechanisms

Mechanisms leading to gene variations are responsible for the diversity of species and are important components of the theory of evolution. One constraint on gene evolution is that of protein foldability; the three-dimensional shapes of proteins must be thermodynamically stable. We explore the impact of this constraint and calculate properties of foldable sequences using 3660 structures from the Protein Data Bank. We seek a selection function that receives sequences as input, and outputs survival probability based on sequence fitness to structure. We compute the number of sequences that match a particular protein structure with energy lower than the native sequence, the density of the number of sequences, the entropy, and the "selection" temperature. The mechanism of structure selection for sequences longer than 200 amino acids is approximately universal. For shorter sequences, it is not. We speculate on concrete evolutionary mechanisms that show this behavior.     

A quantitative investigation of the chemical space surrounding amino acid alphabet formation

To date, explanations for the origin and emergence of the alphabet of amino acids encoded by the standard genetic code have been largely qualitative and speculative. Here, with the help of computational chemistry, we present the first quantitative exploration of nature's “choices” set against various models for plausible alternatives. Specifically, we consider the chemical space defined by three fundamental biophysical properties (size, charge, and hydrophobicity) to ask whether the amino acids that entered the genetic code exhibit a higher diversity than random samples of similar size drawn from several different definitions of amino acid possibility space.We found that in terms of the properties studied, the full, standard set of 20 biologically encoded amino acids is indeed significantly more diverse than an equivalently sized group drawn at random from the set of plausible, prebiotic alternatives (using the Murchison meteorite as a model for pre-biotic plausibility). However, when the set of possible amino acids is enlarged to include those that are produced by standard biosynthetic pathways (reflecting the widespread idea that many members of the standard alphabet were recruited in this way), then the genetically encoded amino acids can no longer be distinguished as more diverse than a random sample. Finally, if we turn to consider the overlap between biologically encoded amino acids and those that are prebiotically plausible, then we find that the biologically encoded subset are no more diverse as a group than would be expected from a random sample, unless the definition of “random sample” is adjusted to reflect possible prebiotic abundance (again, using the contents of the Murchison meteorite as our estimator). This final result is contingent on the accuracy of our computational estimates for amino acid properties, and prebiotic abundances, and an exploration of the likely effect of errors in our estimation reveals that our results should be treated with caution. We thus present this work as a first step in quantifying and thus testing various origin-of-life hypotheses regarding the origin and evolution of life's amino acid alphabet, and advocate the progress that would add valuable information in the future.     

Foldability, enzymatic activity, and interacting ability of barnase mutants obtained by permutation of secondary structure units

Barnase, a well-characterized ribonuclease, has been decomposed into six modules (M1-M6) or secondary structure units (S1-S6). We have studied the foldability and activity of the barnase mutants obtained by permutation of the four internal modules (M2-M5) or secondary structure units (S2-S5) to investigate whether permutation of these building blocks is a useful way to create foldable and/or functional proteins. In this study, we found that one of the secondary structure unit mutants was expressed in Escherichia coli only when His102 was substituted by alanine, which is a catalytic residue of wild-type barnase. This mutant (S2354H102A) had ordered conformations, which unfolded cooperatively during urea-induced unfolding experiments. S2354H102A interacted with other barnase mutants to show a distinct RNase activity, although its own activity was quite weak. This interaction was specific, because S2354H102A interacted with only barnase mutants having His 102 and certain orders of the secondary structure units giving a distinct RNase activity. These results suggest that secondary structure units permuted in barnase mutants maintain their intrinsic "interacting ability" that is used for the folding of wild-type barnase, and the units can form certain conformations that complement those of the appropriate counterparts. Seven of 23 secondary structure unit mutants and only 2 of 23 module mutants had RNase activity. On the basis of the results of analyses of foldability and RNase activity of the mutants performed in this and previous studies, we conclude that secondary structure units are more suitable than modules as building blocks to create novel foldable and/or functional proteins in the case of barnase.     

Effect of alphabet size and foldability requirements on protein structure designability.

A number of investigators have addressed the issue of why certain protein structures are especially common by considering structure designability, defined as the number of sequences that would successfully fold into any particular native structure. One such approach, based on foldability, suggested that structures could be classified according to their maximum possible foldability and that this optimal foldability would be highly correlated with structure designability. Other approaches have focused on computing the designability of lattice proteins written with reduced two-letter amino acid alphabets. These different approaches suggested contrasting characteristics of the most designable structures. This report compares the designability of lattice proteins over a wide range of amino acid alphabets and foldability requirements. While all alphabets have a wide distribution of protein designabilities, the form of the distribution depends on how protein "viability" is defined. Furthermore, under increasing foldability requirements, the change in designabilities for all alphabets are in good agreement with the previous conclusions of the foldability approach. Most importantly, it was noticed that those structures that were highly designable for the two-letter amino acid alphabets are not especially designable with higher-letter alphabets.     

Foldability and chameleon propensity of fold‐switching protein sequences

It has been shown recently by Porter & Looger that a significant number of proteins exist that can form more than one stable fold. This note examines the sequences of these fold‐switching proteins by (a) calculating their foldability scores recently introduced by the present author and (b) comparing the propensity of chameleon sequences in fold switchers and in non fold switchers. It has been found that the average foldability score of the fold switchers indicates weaker foldability. As for the propensity of chameleon sequences of length 5 to 7 it was found, somewhat surprisingly, that there is only a very small difference between the fold switchers and the non fold switchers. Furthermore, when looking at amino acid propensities, for several amino acids there was even an opposing trend in the deviation of their propensities from the overall amino acid propensities.     

An RNA foldability metric; implications for the design of rapidly foldable RNA sequences

Evidence is presented suggesting, for the first time, that the protein foldability metric σ = (T_θ − T_f) / T_θ, where T_θ and T_f are, respectively, the collapse and folding transition temperatures, could be used also to measure the foldability of RNA sequences. These results provide further evidence of similarities between the folding energy landscapes of proteins and RNA. The importance of σ is discussed in the context of the in silico design of rapidly foldable RNA sequences.     

Mutational Analysis of the Absolutely Conserved B8Gly： Consequence on Foldability and Activity of Insulin

B8Gly is absolutely conserved in insulins during evolution. Moreover, its corresponding position is always occupied by a Gly residue in other members of insulin superfamily. Previous work showed that Ala replacement of B8Gly significantly decreased both the activity and the foldability of insulin. However, the effects of substitution are complicated, and different replacements sometimes cause significantly different results. To analyze the effects of B8 replacement by different amino acids, three new insulin/single-chain insulin mutants with B8Gly replaced by Ser, Thr or Leu were prepared by protein engineering, and both their foldability and activity were analyzed. In general, replacement of B8Gly by other amino acids causes significant detriment to the foldability of single-chain insulin： the conformations of the three B8 mutants are essentially different from that of wild-type molecules as revealed by circular dichroism; their disulfide stabilities in redox buffer are significantly decreased; their in vitro refolding efficiencies are decreased approximately two folds; the structural stabilities of the mutants with Set or Thr substitution are decreased significantly, while Leu substitution has little effect as measured by equilibrium guanidine denaturation. As far as biological activity is concerned, Ser replacement of B8Gly has only a moderate effect： its insulin receptor-binding activity is 23% of native insulin. But Ttir or Leu replacement produces significant detriment： the receptorbinding potencies of the two mutants are less than 0.2% of native insulin. The present results suggest that Gly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability and activity are concerned.     

Relationships between genomic base content and distribution of mass in coded proteins

The aim of this research was to examine the possible significance of genome/protein relationships in terms of effects on distribution of mass, especially in proteins. Amino acid residues in proteins have side-chains and polypeptide segments. We use "SCM" (side-chain mass), "MCM" (main-chain mass), and "deltaM" (SCM-MCM) as the deviation from "mass balance." Total MCM of the 61 amino acids in the standard code, 3412, equals total SCM: they form a mass balanced set (mean deltaM = 0). Of 14 natural variants of the code, seven have slightly positive mean deltaM values and seven have slightly negative values. Codes with the standard amino acids assigned randomly to the 20 codon sets of the standard code have about one chance in 3,300 of producing a mass balanced set. In natural proteins, as %A + T increases, the proportion of the mass in the side-chains also increases, by about half the amount calculated for standard genes with various AT/GC ratios, partly due to selection of codons with greater variability in composition at synonymous sites. For 203 representative species (including organelles), the total protein mass is distributed approximately equally between SCM and MCM (overall mean deltaM/amino acid residue, -0.06). The attainment of some overall macromolecular mass balance may have been a criterion for selecting the codon/amino acid pairs. When both structural and dynamic requirements are considered, a genetic code based on hydrophobicity and mass balance as key properties seems likely.     

Key elements for protein foldability revealed by a combinatorial approach among similarly folded but distantly related proteins

We have determined the key regions for protein foldability by creating multiple crossover libraries from two proteins that share similar fold but have low sequence identity and differ significantly in stability. One protein is the propeptide of a serine protease, subtilisin BPN', and the other is Pleurotus ostreatus proteinase A inhibitor 1 (POIA1). The propeptide has a compact structure when complexed with subtilisin but is unstructured when isolated, whereas POIA1 takes a stable structure. We selected four of the conserved amino acid residues for the boundaries of crossover sites and utilized these residues to make same cohesive-ends to assemble synthetic DNA fragments. Each segment has one or two secondary structure units, and the interchange of these structural elements produces 32 (= 2(5)) combinations, including the propeptide and POIA1. The stability of these mutants was first screened by formation of turbid zones on skim milk plates containing subtilisin BPN'. It was shown that six variants were foldable and structural units necessary for folding were identified. Further fragmentation and recombination of these mutants (the "multisection" method) revealed that two interactions between secondary structures are important; one is interaction between the loop-alpha1 and beta2-turn-beta3, and the other is hydrophobic interaction between the adjoining beta1 and beta4 strands. We were also able to specify the significant amino acid combinations for tolerance to proteolysis. These combinatorial methods not only elucidate how domains can be interchanged to make the whole protein foldable but also extract essential regions for the function, which is correlated with the instability of the molecule.     

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Probing the interactions between the folding elements early in the folding of Escherichia coli dihydrofolate reductase by systematic sequence perturbation analysis

One of the necessary conditions for a protein to be foldable is the presence of a complete set of folding elements (FEs) that are short contiguous peptide segments distributed over an amino acid sequence. Previous studies indicated the FE assembly model of protein folding, in which the FEs interact with each other and coalesce to form an intermediate(s) early in the folding reaction. This suggests that a clue to the understanding of the determinants of protein foldability can be found by investigating how the FEs interact with each other early in the folding and thereby elucidating roles of the FEs in protein folding. To reveal the formation process of FE-FE interactions, we studied the early folding events of Escherichia coli dihydrofolate reductase (DHFR) utilizing systematic sequence perturbation analysis. Here, systematic single amino acid substitutions were introduced inside of the FEs (W30X in FE2, V40X in FE3, N59X in FE4, and I155X in FE10; X refers to various amino acid residues), and their kinetic refolding reactions were measured by stopped-flow circular dichroism and fluorescence. We show that the interactions around Trp30 and Ile155 are formed in the burst phase intermediate, while those around Val40 and Asn59 are formed in the transition state of the subsequent folding phase (tau5-phase) and in much later processes, respectively. These and previous results suggest that FE2 and FE10, and also FE1 and FE7, involved in the loop subdomain of DHFR, interact with each other within a millisecond time range, while the stable FE3-FE4 interactions are formed in the later processes. This may highlight the important roles of the FEs mainly inside of the loop subdomain in formation of the burst phase intermediate having a hydrophobic cluster and native-like overall topology and in acquisition of the foldability of DHFR.     

The origin of the biologically coded amino acids

Biology uses essentially 20 amino acids for its coded protein enzymes, representing a very small subset of the structurally possible set. Most models of the origin of life suggest organisms developed from environmentally available organic compounds. A variety of amino acids are easily produced under conditions which were believed to have existed on the primitive Earth or in the early solar nebula. The types of amino acids produced depend on the conditions which prevailed at the time of synthesis, which remain controversial. The selection of the biological set is likely due to chemical and early biological evolution acting on the environmentally available compounds based on their chemical properties. Once life arose, selection would have proceeded based on the functional utility of amino acids coupled with their accessibility by primitive metabolism and their compatibility with other biochemical processes. Some possible mechanisms by which the modern set of 20 amino acids was selected starting from prebiotic chemistry are discussed.     

Simplified amino acid alphabets for protein fold recognition and implications for folding

Protein design experiments have shown that the use of specific subsets of amino acids can produce foldable proteins. This prompts the question of whether there is a minimal amino acid alphabet which could be used to fold all proteins. In this work we make an analogy between sequence patterns which produce foldable sequences and those which make it possible to detect structural homologs by aligning sequences, and use it to suggest the possible size of such a reduced alphabet. We estimate that reduced alphabets containing 10-12 letters can be used to design foldable sequences for a large number of protein families. This estimate is based on the observation that there is little loss of the information necessary to pick out structural homologs in a clustered protein sequence database when a suitable reduction of the amino acid alphabet from 20 to 10 letters is made, but that this information is rapidly degraded when further reductions in the alphabet are made.     

Cloning and nucleotide sequence of β-mannanase and cellulase genes from Bacillus sp. 5H

The two genes for -mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM 109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the -mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of M_r 40,803 Da (-mannanase) and 55,420 Da (cellulase). The deduced primary structure of -mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues.     

Measurement of total protein in plant samples in the presence of tannins

A method for measuring total protein in situ in plant samples has been developed using the determination of amino acids released by acid hydrolysis of dried plant material. Standard proteins and plant samples were hydrolyzed with 3% sulfuric acid at 100 degrees C for 24 h and the amino acids released were measured with ninhydrin. Unhydrolyzed plant extracts were also analyzed for free amino acids with ninhydrin. Total amino acid equivalents (protein plus free amino acids) of a diverse set of plant samples was significantly correlated with total protein as estimated by elemental analysis (N X 6.25). The Lowry method as modified by precipitation of proteins with trichloroacetic acid was found to be unsatisfactory for dried plant samples due to the incomplete extractability of proteins. Although some alkaloids caused increased absorbance with ninhydrin, interference with quantification of protein is likely to be minimal. Tannins interfered with the Lowry and Bradford methods but not the ninhydrin method.     

On the origin of the translation system and the genetic code in the RNA world by means of natural selection, exaptation, and subfunctionalization

Background  

The origin of the translation system is, arguably, the central and the hardest problem in the study of the origin of life, and one of the hardest in all evolutionary biology. The problem has a clear catch-22 aspect: high translation fidelity hardly can be achieved without a complex, highly evolved set of RNAs and proteins but an elaborate protein machinery could not evolve without an accurate translation system. The origin of the genetic code and whether it evolved on the basis of a stereochemical correspondence between amino acids and their cognate codons (or anticodons), through selectional optimization of the code vocabulary, as a "frozen accident" or via a combination of all these routes is another wide open problem despite extensive theoretical and experimental studies. Here we combine the results of comparative genomics of translation system components, data on interaction of amino acids with their cognate codons and anticodons, and data on catalytic activities of ribozymes to develop conceptual models for the origins of the translation system and the genetic code.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Theβ-sheets of proteins,the biosynthetic relationships between amino acids,and the origin of the genetic code

Two forces are generally hypothesised as being responsible for conditioning the origin of the organization of the genetic code: the physicochemical properties of amino acids and their biosynthetic relationships (relationships between precursor and product amino acids). If we assume that the biosynthetic relationships between amino acids were fundamental in defining the genetic code, then it is reasonable to expect that the distribution of physicochemical properties among the amino acids in precursor-product relationships cannot be random but must, rather, be affected by some selective constraints imposed by the structure of primitive proteins. Analysis shows that measurements representing the size of amino acids, e.g. bulkiness, are specifically associated to the pairs of amino acids in precursor-product relationships. However, the size of amino acids cannot have been selected per se but, rather, because it reflects the-sheets of proteins which are, therefore, identified as the main adaptive theme promoting the origin of genetic code organization. Whereas there are no traces of the-helix in the genetic code table.The above considerations make it necessary to re-examine the relationship linking the hydrophilicity of the dinucleoside monophosphates of anticodons and the polarity and bulkiness of amino acids. It can be concluded that this relationship seems to be meaningful only between the hydrophilicity of anticodons and the polarity of amino acids. The latter relationship is supposed to have been operative on hairpin structures, ancestors of the tRNA molecule. Moreover, it is on these very structures that the biosynthetic links between precursor and product amino acids might have been achieved, and the interaction between the hydrophilicity of anticodons and the polarity of amino acids might have had a role in the concession of codons (anticodons) from precursors to products.     

The scenario on the origin of translation in the RNA world: in principle of replication parsimony

Background  

It is now believed that in the origin of life, proteins should have been "invented" in an RNA world. However, due to the complexity of a possible RNA-based proto-translation system, this evolving process seems quite complicated and the associated scenario remains very blurry. Considering that RNA can bind amino acids with specificity, it has been reasonably supposed that initial peptides might have been synthesized on "RNA templates" containing multiple amino acid binding sites. This "Direct RNA Template (DRT)" mechanism is attractive because it should be the simplest mechanism for RNA to synthesize peptides, thus very likely to have been adopted initially in the RNA world. Then, how this mechanism could develop into a proto-translation system mechanism is an interesting problem.