Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

AbstractWe have developed a new method for the C-terminus-specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C-terminus of mature proteins in cell-free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50–95%. Here we demonstrate a protein-protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy

In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro

AbstractAdequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of ‘in vitro virus’ was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was ∼10%, thus indicating a population of the in vitro virus to have ∼1012 protein variants, this number being 104 that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions

The Hayabusa Spacecraft Asteroid Multi-Band Imaging Camera: AMICA

The Hayabusa Spacecraft Asteroid Multiband Imaging Camera (AMICA) has acquired more than 1400 multispectral and high-resolution images of its target asteroid, 25143 Itokawa, since late August 2005. In this paper, we summarize the design and performance of AMICA. In addition, we describe the calibration methods, assumptions, and models, based on measurements. Major calibration steps include corrections for linearity and modeling and subtraction of bias, dark current, read-out smear, and pixel-to-pixel responsivity variations. AMICA v-band data were calibrated to radiance using in-flight stellar observations. The other band data were calibrated to reflectance by comparing them to ground-based observations to avoid the uncertainty of the solar irradiation in those bands. We found that the AMICA signal was linear with respect to the input signal to an accuracy of << 1% when the signal level was < 3800 DN. We verified that the absolute radiance calibration of the AMICA v-band (0.55 micron) was accurate to 4% or less, the accuracy of the disk-integrated spectra with respect to the AMICA v-band was about 1%, and the pixel-to-pixel responsivity (flatfield) variation was 3% or less. The uncertainty in background zero-level was 5 DN. From wide-band observations of star clusters, we found that the AMICA optics have an effective focal length of 120.80 \pm 0.03 mm, yielding a field-of-view (FOV) of 5.83 deg x 5.69 deg. The resulting geometric distortion model was accurate to within a third of a pixel. We demonstrated an image-restoration technique using the point-spread functions of stars, and confirmed that the technique functions well in all loss-less images. An artifact not corrected by this calibration is scattered light associated with bright disks in the FOV.Comment: 107 pages, 22 figures, 9 tables. will appear in Icaru

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

A Comprehensive Resource of Interacting Protein Regions for Refining Human Transcription Factor Networks

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs) suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs) displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function