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TitleA General Small-Angle X-ray Scattering-Based Screening Protocol Validated for Protein-RNA Interactions.
Journal, issue, pagesACS Comb Sci, Vol. 20, Issue 4, Page 197-202, Year 2018
Publish dateApr 9, 2018
AuthorsPo-Chia Chen / Pawel Masiewicz / Vladimir Rybin / Dmitri Svergun / Janosch Hennig /
PubMed AbstractWe present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural information on biomolecular interactions independent of prior knowledge, so as to complement ...We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural information on biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pairwise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl). The screening of 35 RNA motifs between 4 to 10 nucleotides reveals a strong variation of resulting complexes, revealed to be preference-switching between 1:1 and 2:2 binding stoichiometries upon addition of structural modeling. Validation of select sequences in isothermal calorimetry and NMR titration retrieves domain-specific roles and function of a guanine anchor. These findings reinforce the suitability of SAXS as a complement in lead identification.
External linksACS Comb Sci / PubMed:29553252
MethodsSAS (X-ray synchrotron)
Structure data

SASDD25:
1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8
Method: SAXS/SANS

SASDDX4: Wild-type Protein sex-lethal (Protein sex-lethal, Sxl)
Method: SAXS/SANS

SASDDY4:
Protein sex-lethal mutant with 10GS-linker (Protein sex-lethal mutant, Sxl10GS)
Method: SAXS/SANS

SASDDZ4:
1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide U8GU
Method: SAXS/SANS

Source
  • Drosophila melanogaster (fruit fly)
  • Synthetic construct (others)

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