The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein–protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation.

A whole-genome phylogeny of the Escherichia coli/Shigella group was constructed by using the feature frequency profile (FFP) method. This alignment-free approach uses the frequencies of l-mer features of whole genomes to infer phylogenic distances. We present two phylogenies that accentuate different aspects of E. coli/Shigella genomic evolution: (i) one based on the compositions of all possible features of length l = 24 (∼8.4 million features), which are likely to reveal the phenetic grouping and relationship among the organisms and (ii) the other based on the compositions of core features with low frequency and low variability (∼0.56 million features), which account for ∼69% of all commonly shared features among 38 taxa examined and are likely to have genome-wide lineal evolutionary signal. Shigella appears as a single clade when all possible features are used without filtering of noncore features. However, results using core features show that Shigella consists of at least two distantly related subclades, implying that the subclades evolved into a single clade because of a high degree of convergence influenced by mobile genetic elements and niche adaptation. In both FFP trees, the basal group of the E. coli/Shigella phylogeny is the B2 phylogroup, which contains primarily uropathogenic strains, suggesting that the E. coli/Shigella ancestor was likely a facultative or opportunistic pathogen. The extant commensal strains diverged relatively late and appear to be the result of reductive evolution of genomes. We also identify clade distinguishing features and their associated genomic regions within each phylogroup. Such features may provide useful information for understanding evolution of the groups and for quick diagnostic identification of each phylogroup.

Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca2+ homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium.

We present a whole-proteome phylogeny of prokaryotes constructed by comparing feature frequency profiles (FFPs) of whole proteomes. Features are l-mers of amino acids, and each organism is represented by a profile of frequencies of all features. The selection of feature length is critical in the FFP method, and we have developed a procedure for identifying the optimal feature lengths for inferring the phylogeny of prokaryotes, strictly speaking, a proteome phylogeny. Our FFP trees are constructed with whole proteomes of 884 prokaryotes, 16 unicellular eukaryotes, and 2 random sequences. To highlight the branching order of major groups, we present a simplified proteome FFP tree of monophyletic class or phylum with branch support. In our whole-proteome FFP trees (i) Archaea, Bacteria, Eukaryota, and a random sequence outgroup are clearly separated; (ii) Archaea and Bacteria form a sister group when rooted with random sequences; (iii) Planctomycetes, which possesses an intracellular membrane compartment, is placed at the basal position of the Bacteria domain; (iv) almost all groups are monophyletic in prokaryotes at most taxonomic levels, but many differences in the branching order of major groups are observed between our proteome FFP tree and trees built with other methods; and (v) previously “unclassified” genomes may be assigned to the most likely taxa. We describe notable similarities and differences between our FFP trees and those based on other methods in grouping and phylogeny of prokaryotes.

Ten complete mammalian genome sequences were compared by using the “feature frequency profile” (FFP) method of alignment-free comparison. This comparison technique reveals that the whole nongenic portion of mammalian genomes contains evolutionary information that is similar to their genic counterparts—the intron and exon regions. We partitioned the complete genomes of mammals (such as human, chimp, horse, and mouse) into their constituent nongenic, intronic, and exonic components. Phylogenic species trees were constructed for each individual component class of genome sequence data as well as the whole genomes by using standard tree-building algorithms with FFP distances. The phylogenies of the whole genomes and each of the component classes (exonic, intronic, and nongenic regions) have similar topologies, within the optimal feature length range, and all agree well with the evolutionary phylogeny based on a recent large dataset, multispecies, and multigene-based alignment. In the strictest sense, the FFP-based trees are genome phylogenies, not species phylogenies. However, the species phylogeny is highly related to the whole-genome phylogeny. Furthermore, our results reveal that the footprints of evolutionary history are spread throughout the entire length of the whole genome of an organism and are not limited to genes, introns, or short, highly conserved, nongenic sequences that can be adversely affected by factors (such as a choice of sequences, homoplasy, and different mutation rates) resulting in inconsistent species phylogenies.

The vast sequence divergence among different virus groups has presented a great challenge to alignment-based sequence comparison among different virus families. Using an alignment-free comparison method, we construct the whole-proteome phylogeny for a population of viruses from 11 viral families comprising 142 large dsDNA eukaryote viruses. The method is based on the feature frequency profiles (FFP), where the length of the feature (l-mer) is selected to be optimal for phylogenomic inference. We observe that (i) the FFP phylogeny segregates the population into clades, the membership of each has remarkable agreement with current classification by the International Committee on the Taxonomy of Viruses, with one exception that the mimivirus joins the phycodnavirus family; (ii) the FFP tree detects potential evolutionary relationships among some viral families; (iii) the relative position of the 3 herpesvirus subfamilies in the FFP tree differs from gene alignment-based analysis; (iv) the FFP tree suggests the taxonomic positions of certain “unclassified” viruses; and (v) the FFP method identifies candidates for horizontal gene transfer between virus families.

For comparison of whole-genome (genic + nongenic) sequences, multiple sequence alignment of a few selected genes is not appropriate. One approach is to use an alignment-free method in which feature (or l-mer) frequency profiles (FFP) of whole genomes are used for comparison—a variation of a text or book comparison method, using word frequency profiles. In this approach it is critical to identify the optimal resolution range of l-mers for the given set of genomes compared. The optimum FFP method is applicable for comparing whole genomes or large genomic regions even when there are no common genes with high homology. We outline the method in 3 stages: (i) We first show how the optimal resolution range can be determined with English books which have been transformed into long character strings by removing all punctuation and spaces. (ii) Next, we test the robustness of the optimized FFP method at the nucleotide level, using a mutation model with a wide range of base substitutions and rearrangements. (iii) Finally, to illustrate the utility of the method, phylogenies are reconstructed from concatenated mammalian intronic genomes; the FFP derived intronic genome topologies for each l within the optimal range are all very similar. The topology agrees with the established mammalian phylogeny revealing that intron regions contain a similar level of phylogenic signal as do coding regions.

Important cellular processes such as cell fate are likely to be controlled by an elaborate orchestration of multiple signaling pathways, many of which are still not well understood or known. Because protein kinases, the members of a large family of proteins involved in modulating many known signaling pathways, are likely to play important roles in balancing multiple signals to modulate cell fate, we focused our initial search for chemical reagents that regulate stem cell fate among known inhibitors of protein kinases. We have screened 41 characterized inhibitors of six major protein kinase subfamilies to alter the orchestration of multiple signaling pathways involved in differentiation of stem cells. We found that some of them cause recognizable changes in the differentiation rates of two types of stem cells, rat mesenchymal stem cells (MSCs) and mouse embryonic stem cells (ESCs). Among many, we describe the two most effective derivatives of the same scaffold compound, isoquinolinesulfonamide, on the stem cell differentiation: rat MSCs to chondrocytes and mouse ESCs to dopaminergic neurons.

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. There have been many convincing evidences for HGT for specific genes or gene families, but there has been no estimate of the global extent of HGT. Here, we present a method of identifying HGT events within a given protein family and estimate the global extent of HGT in all curated protein domain families (≈8,000) listed in the Pfam database. The results suggest four conclusions: (i) for all protein domain families in Pfam, the fixation of genes horizontally transferred is not a rampant phenomenon between organisms with substantial phylogenetic separations (1.1–9.7% of Pfam families surveyed at three taxonomic ranges studied show indication of HGT); (ii) however, at the level of domains, >50% of Archaea have one or more protein domains acquired by HGT, and nearly 30–50% of Bacteria did the same when examined at three taxonomic ranges. But, the equivalent value for Eukarya is <10%; (iii) HGT will have very little impact in the construction of organism phylogeny, when the construction methods use whole genomes, large numbers of common genes, or SSU rRNAs; and (iv) there appears to be no strong preference of HGT for protein families of particular cellular or molecular functions.

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. There have been many convincing evidences for HGT for specific genes or gene families, but there has been no estimate of the global extent of HGT. Here, we present a method of identifying HGT events within a given protein family and estimate the global extent of HGT in all curated protein domain families (≈8,000) listed in the Pfam database. The results suggest four conclusions: (i) for all protein domain families in Pfam, the fixation of genes horizontally transferred is not a rampant phenomenon between organisms with substantial phylogenetic separations (1.1–9.7% of Pfam families surveyed at three taxonomic ranges studied show indication of HGT); (ii) however, at the level of domains, >50% of Archaea have one or more protein domains acquired by HGT, and nearly 30–50% of Bacteria did the same when examined at three taxonomic ranges. But, the equivalent value for Eukarya is <10%; (iii) HGT will have very little impact in the construction of organism phylogeny, when the construction methods use whole genomes, large numbers of common genes, or SSU rRNAs; and (iv) there appears to be no strong preference of HGT for protein families of particular cellular or molecular functions.

Advances in sequence genomics have resulted in an accumulation of a huge number of protein sequences derived from genome sequences. However, the functions of a large portion of them cannot be inferred based on the current methods of sequence homology detection to proteins of known functions. Three-dimensional structure can have an important impact in providing inference of molecular function (physical and chemical function) of a protein of unknown function. Structural genomics centers worldwide have been determining many 3-D structures of the proteins of unknown functions, and possible molecular functions of them have been inferred based on their structures. Combined with bioinformatics and enzymatic assay tools, the successful acceleration of the process of protein structure determination through high throughput pipelines enables the rapid functional annotation of a large fraction of hypothetical proteins. We present a brief summary of the process we used at the Berkeley Structural Genomics Center to infer molecular functions of proteins of unknown function.

We have constructed a map of the “protein structure space” by using the pairwise structural similarity scores calculated for all nonredundant protein structures determined experimentally. As expected, proteins with similar structures clustered together in the map and the overall distribution of structural classes of this map followed closely that of the map of the “protein fold space” we have reported previously. Consequently, proteins sharing similar molecular functions also were found to colocalize in the protein structure space map, pointing toward a previously undescribed scheme for structure-based functional inference for remote homologues based on the proximity in the map of the protein structure space. We found that this scheme consistently outperformed other predictions made by using either the raw scores or normalized Z-scores of pairwise dali structure alignment.

We have mapped protein conformational space from two to seven residue lengths by employing multidimensional scaling on a data matrix composed of pair-wise angular distances for multiple ϕ-Ψ values collected from high-resolution protein structures. The resulting global maps show clustering of peptide conformations that reveals a dramatic reduction of conformational space as sampled by experimentally observed peptides. Each map can be viewed as a higher order ϕ-Ψ plot defining regions of space that are conformationally allowed.

Type I restriction-modification enzymes are differentiated from type II and type III enzymes by their recognition of two specific dsDNA sequences separated by a given spacer and cleaving DNA randomly away from the recognition sites. They are oligomeric proteins formed by three subunits: a specificity subunit, a methylation subunit, and a restriction subunit. We solved the crystal structure of a specificity subunit from Methanococcus jannaschii at 2.4-Å resolution. Two highly conserved regions (CRs) in the middle and at the C terminus form a coiled–coil of long antiparallel α-helices. Two target recognition domains form globular structures with almost identical topologies and two separate DNA binding clefts with a modeled DNA helix axis positioned across the CR helices. The structure suggests that the coiled–coil CRs act as a molecular ruler for the separation between two recognized DNA sequences. Furthermore, the relative orientation of the two DNA binding clefts suggests kinking of bound dsDNA and exposing of target adenines from the recognized DNA sequences.

Measures of structural similarity between known protein structures provide an objective basis for classifying protein folds and for revealing a global view of the protein structure universe. Here, we describe a rapid method to measure structural similarity based on the profiles of representative local features of Cα distance matrices of compared protein structures. We first extract a finite number of representative local feature (LF) patterns from the distance matrices of all protein fold families by medoid analysis. Then, each Cα distance matrix of a protein structure is encoded by labeling all its submatrices by the index of the nearest representative LF patterns. Finally, the structure is represented by the frequency distribution of these indices, which we call the LF frequency (LFF) profile of the protein. The LFF profile allows one to calculate structural similarity scores among a large number of protein structures quickly, and also to construct and update the “map” of the protein structure universe easily. The LFF profile method efficiently maps complex protein structures into a common Euclidean space without prior assignment of secondary structure information or structural alignment.

The small heat-shock protein (sHSP) from Methanococcus jannaschii (Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of ≈120 Å and an internal diameter of ≈65 Å with six square “windows” of ≈17 Å across and eight triangular windows of ≈30 Å across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified single-chain monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80°C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80°C, but substantial conformational changes of the substrate are required for complex formation at 80°C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by “preassembly condensation” of multiple monomers before final assembly of the sphere.

We describe a procedure for predicting the tertiary folds of α-helical proteins from their primary sequences. The central component of the procedure is a method for predicting interhelical contacts that is based on a helix-packing model. Instead of predicting the individual contacts, our method attempts to identify the entire patch of contacts that involve residues regularly spaced in the sequences. We use this component to glue together two powerful existing methods: a secondary structure prediction program, whose output serves as the input to the contact prediction algorithm, and the tortion angle dynamics program, which uses the predicted tertiary contacts and secondary structural states to assemble three-dimensional structures. In the final step, the procedure uses the initial set of simulated structures to refine the predicted contacts for a new round of structure calculation. When tested against 24 small to medium-sized proteins representing a wide range of helical folds, the completely automated procedure is able to generate native-like models within a limited number of trials consistently.

Bacterial chemotaxis receptors can detect a small concentration gradient of attractants and repellents in the environment over a wide range of background concentration. The clustering of these receptors to form patches observed in vivo and in vitro has been suspected as a reason for the high sensitivity, and such wide dynamic range is thought to be due to the resetting of the receptor sensitivity threshold by methylation/demethylation of the receptors. However, the mechanisms by which such high sensitivity is achieved and how the methylation/demethylation resets the sensitivity are not well understood. A molecular modeling of an intact bacterial chemotaxis receptor based on the crystal structures of a cytoplasmic domain and a periplasmic domain suggests an interesting clustering of three dimeric receptors and a two-dimensional, close-packed lattice formation of the clusters, where each receptor dimer contacts two other receptor dimers at the cytoplasmic domain and two yet different receptor dimers at the periplasmic domain. This interconnection of the receptors to form a patch of receptor clusters suggests a structural basis for the high sensitivity of the bacterial chemotaxis receptors. Furthermore, we present crystallographic data suggesting that, in contrast to most molecular signaling by conformational changes and/or oligomerization of the signaling molecules, the changes in dynamic property of the receptors on ligand binding or methylation may be the language of the signaling by the chemotaxis receptors. Taken together, the changes of the dynamic property of one receptor propagating mechanically to many others in the receptor patch provides a plausible, simple mechanism for the high sensitivity and the dynamic range of the receptors.

The crystal structure of a conserved hypothetical protein, Aq1575, from Aquifex aeolicus has been determined by using x-ray crystallography. The protein belongs to the domain of unknown function DUF28 in the Pfam and PALI databases for which there was no structural information available until now. A structural homology search with the DALI algorithm indicates that this protein has a new fold with no obvious similarity to those of other proteins of known three-dimensional structure. The protein reveals a monomer consisting of three domains arranged along a pseudo threefold symmetry axis. There is a large cleft with approximate dimensions of 10 Å × 10 Å × 20 Å in the center of the three domains along the symmetry axis. Two possible active sites are suggested based on the structure and multiple sequence alignment. There are several highly conserved residues in these putative active sites. The structure based molecular properties and thermostability of the protein are discussed.

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-Å resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.

We examine the interactions between amino acid residues in the context of their secondary structural environments (helix, strand, and coil) in proteins. Effective contact energies for an expanded 60-residue alphabet (20 aa × three secondary structural states) are estimated from the residue–residue contacts observed in known protein structures. Similar to the prototypical contact energies for 20 aa, the newly derived energy parameters reflect mainly the hydrophobic interactions; however, the relative strength of such interactions shows a strong dependence on the secondary structural environment, with nonlocal interactions in β-sheet structures and α-helical structures dominating the energy table. Environment-dependent residue contact energies outperform existing residue pair potentials in both threading and three-dimensional contact prediction tests and should be generally applicable to protein structure prediction.

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies, respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114-kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined. Analysis of the amino acid sequences of AcrB and its homologs revealed the presence of conserved residues at the N-terminus including two phenylalanines which may be exposed to the cytoplasm. Any potential role that these conserved residues may play in function has not been addressed by existing biochemical or structural studies. Since phenylalanine residues elsewhere in the protein have been implicated in ligand binding, we explored the structure of this N-terminal region to investigate structural determinants near the cytoplasmic opening that may mediate drug uptake. Our structure of AcrB in R32 space group reveals an N-terminus loop, reducing the diameter of the central opening to ∼15 Å as opposed to the previously reported value of ∼30 Å for crystal structures in this space group with disordered N-terminus. Recent structures of the AcrB in C2 space group have revealed a helical conformation of this N-terminus but have not discussed its possible implications. We present the crystal structure of AcrB that reveals the structure of the N-terminus containing the conserved residues. We hope that the structural information provides a structural basis for others to design further biochemical investigation of the role of this portion of AcrB in mediating cytoplasmic ligand discrimination and uptake.

The Brn-5 protein, highly expressed in human brain, belongs to the POU family; a class of transcription factors involved in a wide variety of biological processes ranging from programming of embryonic stem cells to cellular housekeeping. This functional diversity is conferred by two DNA-binding subdomains that can assume several configurations due to a bipartite arrangement of POU-specific (POUS) and POU-homeo (POUH) subdomains separated by a linker region. The crystal structure of human Brn-5 transcription factor in complex with corticotrophin-releasing hormone (CRH) gene promoter reveals an unexpected recognition mode of the protein to its cognate DNA. Moreover, the structure also shows the role of the linker in allowing diverse configurations that can be assumed by the two subdomains.