Computational protocol: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

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Protocol publication

[…] NMR spectra were collected at 298 K using either Varian INOVA 600 MHz or INOVA 800 MHz NMR spectrometers (Agilent, Santa Clara, CA, USA), both equipped with cryogenically cooled 1H, 13C, 15N triple-resonance probeheads with z-gradient coils. For 1H NMR spectra, measured at 600 MHz, the recombinant BilRI was diluted in 95%/5% H2O/D2O, 50 mM NaCl, pH 7 buffer in a Shigemi microcell (250 μL). The final BilRI concentration was 4.6 mM. The 1H spectrum was sampled with 20,438 complex points using 64 transients per free induction decay (FID), resulting in an acquisition time of 500 ms in the 1H dimension. The two-dimensional 1H-15N HSQC spectrum of BilRI at pH 5 was measured at the 800 MHz 1H frequency using 128 and 852 complex points in 15N and 1H dimensions, corresponding to acquisition times of 49 ms and 85.2 ms, respectively. A total of 256 transients per FID were used to assure sufficient signal accumulation. The total experimental time was 18 h. Spectra were processed with VnmrJ (Agilent, Santa Clara, CA, USA) and analyzed with Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco, CA, USA) software packages. [...] The binding of recombinant BilRI to various cytokines was analyzed through related-samples Friedman's 2-way analysis of variance on ranks, and this was followed by an analysis of BilRI binding to IL-8 through a paired-samples T-test (IBM SPSS Statistics 22). Due to the small sample size, which was always less than 10, the differences in the biofilm composition, binding capacities and uptake efficiencies of various cytokines of wild-type and bilRI−mutant strains were analyzed using the nonparametric Mann-Whitney U-test (IBM SPSS Statistics 22). The effects of cytokines on the biofilm amount and composition were analyzed using a Kruskall-Wallis test followed by paired Mann-Whitney U-tests with Bonferroni corrections (IBM SPSS Statistics 22) when needed. Differences were regarded as statistically significant at p < 0.05. […]

Pipeline specifications

Software tools Sparky, SPSS
Applications Miscellaneous, NMR-based proteomics analysis
Organisms Aggregatibacter actinomycetemcomitans, Bacteria
Diseases Haemophilus Infections, Neoplasms
Chemicals Amino Acids