Origins of globular structure in proteins

AbstractSince natural proteins are the products of a long evolutionary process, the structural properties of present-day proteins should depend not only on physico-chemical constraints, but also on evolutionary constraints. Here we propose a model for protein evolution, in which membranes play a key role as a scaffold for supporting the gradual evolution from flexible polypeptides to well-folded proteins. We suggest that the folding process of present-day globular proteins is a relic of this putative evolutionary process. To test the hypothesis that membranes once acted as a cradle for the folding of globular proteins, extensive research on membrane proteins and the interactions of globular proteins with membranes will be required

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution

AbstractProtein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 β-lactamase and its inhibitory protein as our model protein-ligand system. ‘Allosteric GFP biosensors’ constructed by this method may be used in a wide range of applications including biochemistry and cell biology

Comparison of the Frequency of Functional SH3 Domains with Different Limited Sets of Amino Acids Using mRNA Display

Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory

In Vitro

In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library

Affinity selection of DNA-binding protein complexes using mRNA display

Comprehensive analysis of DNA–protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes

A Phthalimide Derivative That Inhibits Centrosomal Clustering Is Effective on Multiple Myeloma

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma

Protein–protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein–protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein–protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences

DNA Display Selection of Peptide Ligands for a Full-Length Human G Protein-Coupled Receptor on CHO-K1 Cells

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells