Sequence-dependent Information Research Articles

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

Learning to Accelerate Decomposition for Multi-Directional 3D Printing

Multi-directional 3D printing has the capability of decreasing or eliminating the need for support structures. Recent work proposed a beam-guided search algorithm to find an optimized sequence of plane-clipping, which gives volume decomposition of a given 3D model. Different printing directions are employed in different regions to fabricate a model with tremendously less support (or even no support in many cases). To obtain optimized decomposition, a large beam width needs to be used in the search algorithm, leading to a very time-consuming computation. In this letter, we propose a learning framework that can accelerate the beam-guided search by using a smaller number of the original beam width to obtain results with similar quality. Specifically, we use the results of beam-guided search with large beam width to train a scoring function for candidate clipping planes based on six newly proposed feature metrics. With the help of these feature metrics, both the current and the sequence-dependent information are captured by the neural network to score candidates of clipping. As a result, we can achieve around 3× computational speed. We test and demonstrate our accelerated decomposition on a large dataset of models for 3D printing.

Poly(peptide): Synthesis, Structure, and Function of Peptide-Polymer Amphiphiles and Protein-like Polymers.

In this Account, we describe the organization of functional peptides as densely arrayed side chains on polymer scaffolds which we introduce as a new class of material called poly(peptide). We describe two general classes of poly(peptide): (1) Peptide-Polymer Amphiphiles (PPAs), which consist of block copolymers with a dense grouping of peptides arrayed as the side chains of the hydrophilic block and connected to a hydrophobic block that drives micelle assembly, and (2) Protein-like Polymers (PLPs), wherein peptide-brush polymers are composed from monomers, each containing a peptide side chain. Peptides organized in this manner imbue polymers or polymeric nanoparticles with a range of functional qualities inherent to their specific sequence. Therefore, polymers or nanoparticles otherwise lacking bioactivity or responsiveness to stimuli, once linked to a peptide of choice, can now bind proteins, enter cells and tissues, have controlled and switchable biodistribution patterns, and be enzyme substrates (e.g., for kinases, phosphatases, proteases). Indeed, where peptide substrates are incorporated, kinetically or thermodynamically driven morphological transitions can be enzymatically induced in the polymeric material. Synergistically, the polymer enforces changes in peptide activity and function by virtue of packing and constraining the peptide. The scaffold can protect peptides from proteolysis, change the pharmacokinetic profile of an intravenously injected peptide, increase the cellular uptake of an otherwise cell impermeable therapeutic peptide, or change peptide substrate activity entirely. Moreover, in addition to the sequence-controlled peptides (generated by solid phase synthesis), the polymer can carry its own sequence-dependent information, especially through living polymerization strategies allowing well-defined blocks and terminal labels (e.g., dyes, contrast agents, charged moieties). Hence, the two elements, peptide and polymer, cooperate to yield materials with unique function and properties quite apart from each alone. Herein, we describe the development of synthetic strategies for accessing these classes of biomolecule polymer conjugates. We discuss the utility of poly(peptide)-based materials in a range of biomedical applications, including imaging of diseased tissues (myocardial infarction and cancer), delivering small molecule drugs to tumors with high specificity, imparting cell permeability to otherwise impermeable peptides, protecting bioactive peptides from proteolysis in harsh conditions (e.g., stomach acid and whole blood), and transporting proteins into traditionally difficult-to-transfect cell types, including stem cells. Poly(peptide) materials offer new properties to both the constituent peptides and to the polymers, which can be tuned by the design of the oligopeptide sequence, degree of polymerization, peptide arrangement on the polymer backbone, and polymer backbone chemistry. These properties establish this approach as valuable for the development of peptides as medicines and materials in a range of settings.

Reduced alphabet of prebiotic amino acids optimally encodes the conformational space of diverse extant protein folds

BackgroundThere is wide agreement that only a subset of the twenty standard amino acids existed prebiotically in sufficient concentrations to form functional polypeptides. We ask how this subset, postulated as {A,D,E,G,I,L,P,S,T,V}, could have formed structures stable enough to found metabolic pathways. Inspired by alphabet reduction experiments, we undertook a computational analysis to measure the structural coding behavior of sequences simplified by reduced alphabets. We sought to discern characteristics of the prebiotic set that would endow it with unique properties relevant to structure, stability, and folding.ResultsDrawing on a large dataset of single-domain proteins, we employed an information-theoretic measure to assess how well the prebiotic amino acid set preserves fold information against all other possible ten-amino acid sets. An extensive virtual mutagenesis procedure revealed that the prebiotic set excellently preserves sequence-dependent information regarding both backbone conformation and tertiary contact matrix of proteins. We observed that information retention is fold-class dependent: the prebiotic set sufficiently encodes the structure space of α/β and α + β folds, and to a lesser extent, of all-α and all-β folds. The prebiotic set appeared insufficient to encode the small proteins. Assessing how well the prebiotic set discriminates native vs. incorrect sequence-structure matches, we found that α/β and α + β folds exhibit more pronounced energy gaps with the prebiotic set than with nearly all alternatives.ConclusionsThe prebiotic set optimally encodes local backbone structures that appear in the folded environment and near-optimally encodes the tertiary contact matrix of extant proteins. The fold-class-specific patterns observed from our structural analysis confirm the postulated timeline of fold appearance in proteogenesis derived from proteomic sequence analyses. Polypeptides arising in a prebiotic environment will likely form α/β and α + β-like folds if any at all. We infer that the progressive expansion of the alphabet allowed the increased conformational stability and functional specificity of later folds, including all-α, all-β, and small proteins. Our results suggest that prebiotic sequences are amenable to mutations that significantly lower native conformational energies and increase discrimination amidst incorrect folds. This property may have assisted the genesis of functional proto-enzymes prior to the expansion of the full amino acid alphabet.

Investigating the Formation of Structural Elements in Proteins Using Local Sequence-Dependent Information and a Heuristic Search Algorithm.

Structural elements inserted in proteins are essential to define folding/unfolding mechanisms and partner recognition events governing signaling processes in living organisms. Here, we present an original approach to model the folding mechanism of these structural elements. Our approach is based on the exploitation of local, sequence-dependent structural information encoded in a database of three-residue fragments extracted from a large set of high-resolution experimentally determined protein structures. The computation of conformational transitions leading to the formation of the structural elements is formulated as a discrete path search problem using this database. To solve this problem, we propose a heuristically-guided depth-first search algorithm. The domain-dependent heuristic function aims at minimizing the length of the path in terms of angular distances, while maximizing the local density of the intermediate states, which is related to their probability of existence. We have applied the strategy to two small synthetic polypeptides mimicking two common structural motifs in proteins. The folding mechanisms extracted are very similar to those obtained when using traditional, computationally expensive approaches. These results show that the proposed approach, thanks to its simplicity and computational efficiency, is a promising research direction.

Molecular Dynamics Simulations of the d(CCAACGTTGG) 2 Decamer: Influence of the Crystal Environment

Molecular dynamics (MD) simulations of the DNA duplex d(CCAACGTTGG) 2 were used to study the relationship between DNA sequence and structure. Two crystal simulations were carried out; one consisted of one unit cell containing two duplexes, and the other of two unit cells containing four duplexes. Two solution simulations were also carried out, one starting from canonical B-DNA and the other starting from the crystal structure. For many helicoidal parameters, the results from the crystal and solution simulations were essentially identical. However, for other parameters, in particular, α, γ, δ, (ϵ − ζ), phase, and helical twist, differences between crystal and solution simulations were apparent. Notably, during crystal simulations, values of helical twist remained comparable to those in the crystal structure, to include the sequence-dependent differences among base steps, in which values ranged from 20° to 50° per base step. However, in the solution simulations, not only did the average values of helical twist decrease to ∼30° per base step, but every base step was ∼30°, suggesting that the sequence-dependent information may be lost. This study reveals that MD simulations of the crystal environment complement solution simulations in validating the applicability of MD to the analysis of DNA structure.

Genotypic detection of mutations in rifampicin resistant clinical isolates ofMycobacterium tuberculosis.

The resistance of majority ofM. tuberculosis strains to rifampicin a key drug in the tuberculosis therapy, has been cause of major alarm world-wide. Such an association of strain with the drug takes a long time i.e. 2-3 months. We have modified a method for rapid detection of rifampicin resistant strains by screening them through PCR-SSCP.We have identified mutations in rpo, β region of the gene encoding, β sub-unit of RNA polymerase in rifampicin resistant 67 clinical isolates ofM. tuberculosis. Mutations were screened in these isolates by single strand confirmation polymorphism (SSCP). When DNA sequencing data was compared with the result of SSCP analysis, direct PCR sequencing results were more easily interpreted and contatined more sequence dependent information. These findings provide the basis for rapid detection of rifampicin esistance, a marker of multidrug resistance.

Redox potentials of active-site bis(cysteinyl) fragments of thiol-protein oxidoreductases.

The active sites of thiol-protein oxidoreductases consist of the characteristic Cys-X-X-Cys motif, and the redox potentials of these enzymes reflect the propensity of the bis(cysteinyl) sequence portion for disulfide loop formation. Thereby, as is known from comparing the three-dimensional (3D) structures of thioredoxin and glutaredoxin in the reduced and oxidized state, reduction of the disulfide bond is accompanied by minimal perturbation of the backbone folding of the active sites. In order to estimate the sequence-dependent intrinsic free energy of formation of the active-site disulfide loops in oxidoreductases, synthetic fragments corresponding to the sequences 31-38, 10-17, 134-141, and 34-41 of thioredoxin, glutaredoxin, thioredoxin reductase, and protein disulfide isomerase (PDI), respectively, were analyzed for their tendency to form 14-membered rings. For this purpose thiol/disulfide exchange experiments, with glutathione as reference redox pair, were performed on the bis(cysteinyl) octapeptides. As the free energy of ring closure of linear peptides consists mainly of the free energy of formation of the disulfide loop with a defined geometry from a statistical ensemble of conformations of the bis(cysteinyl) peptides, the observed differences in the equilibrium constants, although relatively small (within a factor 10), suggest that sequence-dependent information for loop formation is retained in the excised active-site fragments. These inherent redox potentials are, however, significantly affected and/or amplified in the native proteins by the conformational restraints imposed by the "structural domains" on the "functional domains".