IPRO: an iterative computational protein library redesign and optimization procedure

A number of computational approaches have been developed to reengineer promising chimeric proteins one at a time through targeted point mutations. In this article, we introduce the computational procedure IPRO (iterative protein redesign and optimization procedure) for the redesign of an entire combinatorial protein library in one step using energy-based scoring functions. IPRO relies on identifying mutations in the parental sequences, which when propagated downstream in the combinatorial library, improve the average quality of the library (e.g., stability, binding affinity, specific activity, etc.). Residue and rotamer design choices are driven by a globally convergent mixed-integer linear programming formulation. Unlike many of the available computational approaches, the procedure allows for backbone movement as well as redocking of the associated ligands after a prespecified number of design iterations. IPRO can also be used, as a limiting case, for the redesign of a single or handful of individual sequences. The application of IPRO is highlighted through the redesign of a 16-member library of Escherichia coli/Bacillus subtilis dihydrofolate reductase hybrids, both individually and through upstream parental sequence redesign, for improving the average binding energy. Computational results demonstrate that it is indeed feasible to improve the overall library quality as exemplified by binding energy scores through targeted mutations in the parental sequences.     

A Review on Epigenetic Inheritance of Experiences in Humans

Biochemical Genetics - If genetics defines the inheritance of DNA, epigenetics aims to regulate and make it adaptable. Epigenetic alterations include DNA methylation, chromatin remodelling,...     

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by a 951?bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5′ and 3′ splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522?nt), separating the catalytic core from the 3′ splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3′ splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Nek2B stimulates zygotic centrosome assembly in Xenopus laevis in a kinase-independent manner

Pronuclear migration and formation of the first mitotic spindle depend upon assembly of a functional zygotic centrosome. For most animals, this involves both paternal and maternal contributions as sperm basal bodies are converted into centrosomes competent for microtubule nucleation through recruitment of egg proteins. Nek2B is a vertebrate NIMA-related protein kinase required for centrosome assembly, as its depletion from egg extracts delays microtubule aster formation from sperm basal bodies. Using Xenopus as a model system, we now show that protein expression of Nek2B begins during mid-oogenesis and increases further upon oocyte maturation. This is regulated, at least in part, at the level of protein translation. Nek2B protein is weakly phosphorylated in mitotic egg extracts but its recruitment to the sperm basal body, which occurs independently of its kinase activity, stimulates its phosphorylation, possibly through sequestration from a phosphatase present in mitotic egg cytoplasm. Importantly, although Nek2B is not required to organize acentrosomal microtubule asters, we show that addition of either active or kinase-dead recombinant Nek2B can restore centrosome assembly in a dose-dependent manner to a depleted extract. These results support a model in which maternal Nek2B acts to promote assembly of a functional zygotic centrosome in a kinase-independent manner.     

Bax forms two types of channels, one of which is voltage-gated

When activated, the proapoptotic protein Bax permeabilizes the mitochondrial outer membrane, allowing the release of proteins into the cytosol and thus initiating the execution phase of apoptosis. When activated Bax was reconstituted into phospholipid membranes, we discovered a new, to our knowledge, property of Bax channels: voltage gating. We also found that the same Bax sample under the same experimental conditions could give rise to two radically different channels: Type A, which is small, well behaved, homogeneous, and voltage-gated, and Type B, which is large, noisy, and voltage-independent. One Type B channel can be converted irreversibly into a population of Type A channels by the addition of La³⁺. This conversion process appears to involve a two-dimensional budding mechanism. The existence of these two types of Bax channels suggests a process for controlling the degree of mitochondrial outer membrane permeabilization.     

Comparison of repetitive-sequence-based polymerase chain reaction with random amplified polymorphic DNA analysis for rapid genotyping of nontuberculosis mycobacteria

Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit?- variable number of tandem repeat (MIRU-VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU-VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.