Guide oligonucleotide-dependent DNA linkage that facilitates controllable polymerization of microgene blocks

Faster and more efficient searches of a huge protein sequence space for the purpose of conducting experiments in protein evolution can be achieved through the development of a block shuffling-based evolution system. One of the key components of such a system is the accurate and efficient linkage of gene units. Here we introduce a new method that allows accurate and controllable linkage of microgene blocks. This method employs a thermostable DNA ligase that links two single-stranded microgene blocks when they hybridize a complementary guide oligonucleotide. At high temperature, the ligation of the microgene units is fully dependent on the guide oligonucleotide, which can exclude undesired polymer formation, including the incorporation of microgenes having illegitimate sizes and "head-to-head" and "tail-to-tail" ligation of blocks. We were also able to assemble three microgene units using two guide oligonucleotides. Using this method of controllable linkage should facilitate further development of a step-by-step system for the polymerization of gene blocks, leading to a versatile block shuffling-based protein evolution system.     

Translated products of tandem microgene repeats exhibit diverse properties also seen in natural proteins

Repetitiousness is often observed in the primary and tertiary structures of proteins. We are intrigued by the potential role played by periodicity in the evolution of proteins and have created artificial repetitious proteins from repeats of short DNA sequences (microgenes). In this paper we characterize the physicochemical properties of six such artificially created proteins, which are the translated products of repeats of three microgenes. Three of the six proteins contain beta-sheet-like structures and are rather hydrophobic in nature. These proteins form macroscopic membranous structures in the presence of monovalent cationic ions, suggesting they have the capacity to promote strong intermolecular interactions. Of the other three proteins, one is comprised of alpha-helices and two have disordered structures. Small angle X-ray scattering analysis indicates that the artificial proteins do not fold as tightly as natural proteins, but are more compact than if completely denatured. One alpha-helical protein whose microgene unit was designed from coiled coil proteins was crystallized, demonstrating that repetitious artificial proteins can undergo transition to a more ordered state under appropriate conditions. Application of this approach to the development of a novel protein engineering system is discussed.     

Combinatorics of peptide sextets encoded by a single microgene

Genetic information stored in DNA sequences is translated into protein by linking a triplet nucleotide sequence and an amino acid. Because the frames of the triplets can be configured in three ways, a total of six polypeptides, each with a different sequence, can be produced from a single double-stranded DNA molecule. We recently developed the MolCraft system [reviewed in K. Shiba, J. Mol. Catal. B 18 (2004) xxx], which enables us to make combinatorial polymers of three peptides translated from one strand of a double-stranded DNA molecule. To explore all the information that a single double-stranded DNA molecule encodes, we have now developed a new system, La-MolCraft, in which all six reading frames encoded by both strands are combinatorially polymerized using loop-mediated isothermal amplification of DNA (LAMP) [Nucl. Acids Res. 28 (2000) E63].     

On the role of periodism in the origin of proteins

Two different views have been proposed for origins of genes (or proteins). One is that primordial genes evolved from random sequences. This view underlies the concept of modern in vitro evolution experiments that functional molecules (even proteins) evolved from random sequence-libraries. On the contrary, the second view reminds that "random sequences" would be an unusual state in which to find RNA or DNA, because it is their inherent nature to yield periodic structures during the course of semi-conservative replication. In this second view, the periodicity of DNA (or RNA) is responsible for emergence of primordial genes. Although recent reports on the variety of periodicities present in proteins, genes and genomes are consistent with the second view, it has yet to be experimentally tested. We assessed the significance of periodicities of DNA in the origin of genes by constructing such periodic DNAs. The results showed that periodic DNA produced ordered proteins at very high rates, which is in contrast to the fact that proteins with random sequences lack secondary structures. We concluded that periodicity played a pivotal role in the origin of many genes. The observation should pave the way for new experimental evolution systems for proteins.     

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.     

Evolutionary aspects of protein structure and folding

Four basic stages of evolution of protein structure are described based on recent work of the authors targeted specifically on reconstruction of the earliest events in the protein evolution. According to this reconstruction, the initial stage of short peptides of, probably, only few amino-acid residues had been followed by formation of closed loops of the size 25-30 residues, which corresponds to the polymer-statistically optimal ring closure size for mixed polypeptide chains. The next stage involved fusion of the respective small linear genes and formation of protein structures consisting of several closed loops of the nearly standard size, up to 4-6 loops (100-200 amino acid residues) in a typical protein fold. The last, modern stage began with combinatorial fusion of the presumably circular 300-600 bp DNA units and, accordingly, formation of multidomain proteins.     

Biomolecular engineering and drug development

Biomolecular engineering is a technology to create novel structures of high-value biomolecules for use in medicine and industry, through the directed alteration of proteins and/or biologically active molecules in living cells to produce a novel biometabolites as well as engineered protein itself. For the development of new drugs by biomolecular engineering, desired biomolecules have to be rationally designed based on their structure-stability/structure-activity relationship, and then screened through well-established mutation and selection program. Over the past decade, there has been significant progress in mutation and selection methodology; DNA shuffling technology mimicking natural evolution for artificial DNA recombination and phage-displayed combinatorial peptide library for rapid selection of proteins expressed from mutated genes. Bioinformatic tools including functional genomics and proteomics have been also developed for the ready access to the information related to the protein-function and genome-protein, leading to the design and identification of new drug targets. Throughout the use of an enormous amount of bioinformatic databases, many protein/peptide drugs and biometabolite molecules have been designed. The candidates of new drugs are monoclonal antibodies, vaccines, enzymes, antibiotics, therapeutic peptides, and so on. Two humanized monoclonal antibodies approved by FDA became the first line of drugs designed by biomolecular engineering approach. They are Herceptin and Synagis, for the treatment of breast cancer and pediatric respiratory syncytial viral infection, respectively. Many more newly engineered biomolecules are under developing for medicinal application. Some clinical trials for therapeutic applications are now in progress, and very positive results are already anticipated.     

Evolutionary Aspects of Protein Structure and Folding

Four basic stages of evolution of protein structure are described, basing on recent work of the authors aimed specifically to reconstruct the earliest events in the protein evolution. According to this reconstruction, the initial stage of short peptides comprising, probably, only a few amino acid residues had been followed by formation of closed loops of 25–30 residues, which corresponds to the polymer-statistically optimal ring closure size for mixed polypeptide chains. The next stage involved fusion of relatively small linear genes and formation of protein structures consisting of several closed loops of a nearly standard size, with 4–6 loops (100–200 amino acid residues) in a typical protein fold. The last, modern stage began with combinatorial fusion of the presumably circular 300–600 bp DNA units and, accordingly, formation of multidomain proteins.     

Protein structure prediction via combinatorial assembly of sub-structural units

Following the hierarchical nature of protein folding, we propose a three-stage scheme for the prediction of a protein structure from its sequence. First, the sequence is cut to fragments that are each assigned a structure. Second, the assigned structures are combinatorially assembled to form the overall 3D organization. Third, highly ranked predicted arrangements are completed and refined. This work focuses on the second stage of this scheme: the combinatorial assembly. We present CombDock, a combinatorial docking algorithm. CombDock gets an ordered set of protein sub-structures and predicts the inter-contacts that define their overall organization. We reduce the combinatorial assembly to a graph-theory problem, and give a heuristic polynomial solution to this computationally hard problem. We applied CombDock to various examples of structural units of two types: protein domains and building blocks, which are relatively stable sub-structures of domains. Moreover, we tested CombDock using increasingly distorted input, where the native structural units were replaced by similarly folded units extracted from homologous proteins and, in the more difficult cases, from globally unrelated proteins. The algorithm is robust, showing low sensitivity to input distortion. This suggests that CombDock is a useful tool in protein structure prediction that may be applied to large target proteins.     

Evolution without speciation but with selection: LUCA, the Last Universal Common Ancestor in Gilbert's RNA world

This is not an attempt to analyze the Last Universal Common Ancestor (LUCA) to understand the origin of living systems. We do not know what came before Gilberts' RNA world. Our analysis starts with the RNA world and with genes (biological replicators alla Dawkings) made up of RNA proteins with enzymatic catalytic functions within units that are not yet modern cells. We offer a scenario where cellular entities are very simple and without individuality; they are only simple primary units of selection (the first level of selection) in which replicators compete in the most Darwinian manner, totally deprived of cooperation and interactions among genes. The information processing system of this RNA world is inaccurate and inefficient when compared to that found in organisms that came later. Among the "genes" and the entities that harbor them, high mutation rate was the most prevalent source of variability and the only inheritance was through lateral gene transfer of mobile elements. There were no chromosomes or any other genomic organization. As millions of years accumulated, complex and organized biological structures and processes evolved thanks to the variability mustered up mostly by lateral gene transfers and mutations. With micro- and mini-satellites, lateral gene transfers became indispensable devices of selection to mold variability. Competition and Darwinian selection gave way to a new transition in evolution, one I consider ineluctable, in which cooperation among interactive genes prevailed for the sake of higher fitness. Compartmentalization constituted a major transition in evolution that spurted new types of genome organization. Minichromosomes is one of these; cellular membranes and cytoplasmic structures completed the picture of the primitive cell. However, the much talked about phylogenetic tree does not exit in that ancient LUCA. The tree has no organism at its base; only clusters of genes evoke a fragile beginning for the increasingly complex cell types that were to emerge later.     

SELEX技术在神经系统疾病研究中的应用

配体指数级富集系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术是一种组合化学技术,可经过反复筛选扩增得到针对靶分子的高亲和力和高特异性的适配子.适配子通过识别、结合特定靶分子并对其进行功能调控从而达到对疾病诊断和治疗的目的 .近年来SELEX技术在神经系统功能和疾病研究中的应用越来越多.现已经筛选出针对朊蛋白、肌腱蛋白-C、β-淀粉样肽、乙酰胆碱受体的自身抗体等靶标的适配子,促进了对朊病毒病、脑肿瘤、阿茨海默病、重症肌无力等神经系统疾病的诊断和治疗研究,为这些疾病的诊治提供了新的研究工具.     

Construction of a random circular permutation library using an engineered transposon

Circular permutation is an important protein engineering tool used to create sequence diversity of a protein by changing its linear order of amino acid sequence. Circular permutation has proven to be effective in the evolution of proteins for desired properties while maintaining similar three-dimensional structures. Due to the lack of a robust design principle guiding the selection of new termini, construction of a combinatorial library is much preferred for comprehensive evaluation of circular permutation. Unfortunately, the conventional methods used to create random circular permutation libraries cause significant sequence modification at new termini of circular permutants. In addition, these methods impose additional limitations by requiring either relatively inefficient blunt-end ligation during library construction or redesign of transposons for tailored expression of circular permutants. In this study, we present the development of an engineered transposon for facile construction of random circular permutation libraries. We provide evidence that minimal modification at the new termini of the random circular permutants is possible with our engineered transposon. In addition, our method enables the use of sticky-end ligation during library construction and provides external tunability for expression of random circular permutants.     

Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ～44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.     

Domain combinations in archaeal, eubacterial and eukaryotic proteomes.

There is a limited repertoire of domain families that are duplicated and combined in different ways to form the set of proteins in a genome. Proteins are gene products, and at the level of genes, duplication, recombination, fusion and fission are the processes that produce new genes. We attempt to gain an overview of these processes by studying the evolutionary units in proteins, domains, in the protein sequences of 40 genomes. The domain and superfamily definitions in the Structural Classification of Proteins Database are used, so that we can view all pairs of adjacent domains in genome sequences in terms of their superfamily combinations. We find 783 out of the 859 superfamilies in SCOP in these genomes, and the 783 families occur in 1307 pairwise combinations. Most families are observed in combination with one or two other families, while a few families are very versatile in their combinatorial behaviour; 209 families do not make combinations with other families. This type of pattern can be described as a scale-free network. We also study the N to C-terminal orientation of domain pairs and domain repeats. The phylogenetic distribution of domain combinations is surveyed, to establish the extent of common and kingdom-specific combinations. Of the kingdom-specific combinations, significantly more combinations consist of families present in all three kingdoms than of families present in one or two kingdoms. Hence, we are led to conclude that recombination between common families, as compared to the invention of new families and recombination among these, has also been a major contribution to the evolution of kingdom-specific and species-specific functions in organisms in all three kingdoms. Finally, we compare the set of the domain combinations in the genomes to those in the RCSB Protein Data Bank, and discuss the implications for structural genomics.     

Convergent evolution with combinatorial peptides

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.     

An insight into domain combinations

Domains are the building blocks of all globular proteins, and are units of compact three-dimensional structure as well as evolutionary units. There is a limited repertoire of domain families, so that these domain families are duplicated and combined in different ways to form the set of proteins in a genome. Proteins are gene products. The processes that produce new genes are duplication and recombination as well as gene fusion and fission. We attempt to gain an overview of these processes by studying the structural domains in the proteins of seven genomes from the three kingdoms of life: Eubacteria, Archaea and Eukaryota. We use here the domain and superfamily definitions in Structural Classification of Proteins Database (SCOP) in order to map pairs of adjacent domains in genome sequences in terms of their superfamily combinations. We find 624 out of the 764 superfamilies in SCOP in these genomes, and the 624 families occur in 585 pairwise combinations. Most families are observed in combination with one or two other families, while a few families are very versatile in their combinatorial behaviour. This type of pattern can be described by a scale-free network. Finally, we study domain repeats and we compare the set of the domain combinations in the genomes to those in PDB, and discuss the implications for structural genomics.     

Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.     

Association of polymorphisms in odorant-binding protein genes with variation in olfactory response to benzaldehyde in Drosophila

Adaptive evolution of animals depends on behaviors that are essential for their survival and reproduction. The olfactory system of Drosophila melanogaster has emerged as one of the best characterized olfactory systems, which in addition to a family of odorant receptors, contains an approximately equal number of odorant-binding proteins (OBPs), encoded by a multigene family of 51 genes. Despite their abundant expression, little is known about their role in chemosensation, largely due to the lack of available mutations in these genes. We capitalized on naturally occurring mutations (polymorphisms) to gain insights into their functions. We analyzed the sequences of 13 Obp genes in two chromosomal clusters in a population of wild-derived inbred lines, and asked whether polymorphisms in these genes are associated with variation in olfactory responsiveness. Four polymorphisms in 3 Obp genes exceeded the statistical permutation threshold for association with responsiveness to benzaldehyde, suggesting redundancy and/or combinatorial recognition by these OBPs of this odorant. Model predictions of alternative pre-mRNA secondary structures associated with polymorphic sites suggest that alterations in Obp mRNA structure could contribute to phenotypic variation in olfactory behavior.     

Combinatorial docking approach for structure prediction of large proteins and multi-molecular assemblies

Protein folding and protein binding are similar processes. In both, structural units combinatorially associate with each other. In the case of folding, we mostly handle relatively small units, building blocks or domains, that are covalently linked. In the case of multi-molecular binding, the subunits are relatively large and are associated only by non-covalent bonds. Experimentally, the difficulty in the determination of the structures of such large assemblies increases with the complex size and the number of components it contains. Computationally, the prediction of the structures of multi-molecular complexes has largely not been addressed, probably owing to the magnitude of the combinatorial complexity of the problem. Current docking algorithms mostly target prediction of pairwise interactions. Here our goal is to predict the structures of multi-unit associations, whether these are chain-connected as in protein folding, or separate disjoint molecules in the assemblies. We assume that the structures of the single units are known, either through experimental determination or modeling. Our aim is to combinatorially assemble these units to predict their structure. To address this problem we have developed CombDock. CombDock is a combinatorial docking algorithm for the structural units assembly problem. Below, we briefly describe the algorithm and present examples of its various applications to folding and to multi-molecular assemblies. To test the robustness of the algorithm, we use inaccurate models of the structural units, derived either from crystal structures of unbound molecules or from modeling of the target sequences. The algorithm has been able to predict near-native arrangements of the input structural units in almost all of the cases, suggesting that a combinatorial approach can overcome the imperfect shape complementarity caused by the inaccuracy of the models. In addition, we further show that through a combinatorial docking strategy it is possible to enhance the predictions of pairwise interactions involved in a multi-molecular assembly.