细胞内蛋白网络通过负性选择达到最佳特异性
本周Nature上一篇文章报道相关内容。细胞内蛋白质网络是非常复杂的，许多种类蛋白参与某一类信号，这些蛋白质如何保持特异性？作者认为在长期进化过程中，经过系统广泛的负性选择使蛋白质间相互作用网络特异性达到最佳化。
| Nature 426, 676 - 680 (11 December 2003); doi:10.1038/nature02178 |
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ALI ZARRINPAR1,2, SANG-HYUN PARK2 & WENDELL A. LIM2
spectrometry. Concentrations were verified by quantitative amino acid analysis.
SH3 domain array binding assay 100 µl each of 0.1 µM purified GST–SH3 fusion proteins in TBST buffer were spotted in array format on prewetted nitrocellulose membrane with a dot-blot apparatus. Array membranes were blocked in 3% milk, 1% BSA in TBST for 1 h at room temperature, and then probed with 6 ml of the His-tagged fusion protein containing the proline-rich peptide of interest (50 µM) in TBST for 4–16 h at 4 °C. The membrane was washed four times in TBST, then reprobed for 1 h with a horseradish peroxidase-conjugated anti-His6 antibody (dilution 1:2000; Santa Cruz Biotechnology) at 4 °C. Finally, the blot was developed with an enhanced chemiluminescence system and quantified on an AlphaInnotech charge-coupled device camera and analytical software. To control for variation in antibody levels and development exposure, standards of a His-tagged protein (100 µl of 100-nM and 10-nM solutions) were directly spotted on the membrane.
Spot intensities were corrected for background and for variations in spotting and exposure (Supplementary Figs S8a, S11). Corrected intensities for each spot are given relative to the intensity for the Sho1 SH3-domain spot probed with wild-type peptide. The semiquantitative nature of this assay was validated by comparing spot intensities from the SH3-domain arrays with dissociation constants measured in vitro (Supplementary Fig. S8b).
Measurement of binding affinities Affinities of synthetic peptide ligands for SH3 domains were measured by following the increase in domain tryptophan fluorescence on titration of ligand into a solution of SH3 domain at a fixed concentration of 0.01–0.5 µM (always less than 0.25Kd)28. The ligand stock concentration was typically between 0.1 and 2 mM. Data were fitted to the equation
by nonlinear least-squares analysis with the program ProFit 5.6.4 (Quantum Soft), where y is the fluorescence reading, x is ligand concentration, Kd is dissociation constant, F0 is initial fluorescence value (fraction bound = 0) and Fmax is fluorescence value at saturation (fraction bound = 1).
Competition growths Starter cultures of the three strains (Supplementary Fig. S6) were grown independently to an OD600 of 0.5. Equal amounts of each were combined into one tube. An aliquot was removed from this tube as a standard for comparison with subsequent time samples. Cells were diluted 1:100 into various media and incubated at the appropriate temperature until OD600 was 0.5, whereupon they were diluted 1:100 (about once or twice a day). Samples were removed at various time points and lysed by incubation with Zymolyase and boiling. The lysates were subjected to PCR. The PCR product was sequenced in accordance with the standard protocol provided by Applied Biosystems and analysed on an ABI Prism 3700 DNA Analyzer with DNA Sequencing Analysis Software Version 3.6.1 (Applied Biosystems, Foster City, California). Mutant frequencies within the culture were estimated with the sequencing-based protocol developed by Kwok and Duan29. Data were fitted to exponential equations that accounted for changes in growth of the mutant of interest and changes in growth of competitors.
Supplementary information accompanies this paper.
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