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Crystallographic Structural Elucidation of E. coli HU and a Four‐Way Junction
Author(s) -
Khun Charya,
Mukerji Ishita
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.775.5
Subject(s) - crystallography , dna , förster resonance energy transfer , nuclease , chemistry , crystallization , dodecameric protein , biophysics , biology , biochemistry , physics , fluorescence , organic chemistry , quantum mechanics
E. coli histone‐like HU protein binds with high affinity to DNA recombination intermediates, such as the Holliday junction, to facilitate prokaryotic DNA transcription, compaction, recombination, and replication. While previous F örster resonance energy transfer (FRET) studies have determined possible structural models of HU‐junction interaction, X‐ray crystallographic data is needed to confirm those models. Studies have shown that crystals can be obtained with a dodecamer junction complex. However, we believe that HU can also be successfully crystallized with J20, that is, a junction with 20 base pairs per strand. We believe that 20 base pairs per arm is sufficient in limiting the flexibility of our junction. Our investigation aims to determine the most favorable conditions for HU‐J20 crystallization by using various solvents, pH, and viscosities. In our study, we show that our HU samples are pure, nuclease‐free, and bind with high affinity to J20. In addition, we show that our J20 samples are at least 90% properly annealed via a non‐denaturing polyacrylamide gel. Moreover, we have observed the formation of small crystals in a few of our wells, primarily around pH 8.5 in 20% polyethene glycol with 0.2 M ammonium chloride, and 0.005 M calcium dichloride. Going forward, we will screen around our successful conditions with higher protein concentrations. We will also use magnesium dichloride solutions in addition to calcium dichloride solutions, as previous studies have shown that magnesium improves the stabilization of DNA. Upon the development of crystals, we hope to solve the structure of HU‐J20 via diffraction. Support or Funding Information American Society of Biochemistry and Molecular Biology Undergraduate Research Award, Wesleyan Math and Science Scholars Program, Wesleyan University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .