Synthesis of the basic‐helix‐loop‐helix region of the immunoglobulin enhancer binding protein E47 and evaluation of its structural and DNA binding properties

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Abstract
The basic‐helix‐loop‐helix (bHLH) region of the immunoglobulin enhancer binding protein E47 (IEB E47) was prepared in high yield by a solid‐phase peptide synthesis methodology. Size‐exclusion chromatography, sedimentation equilibrium and cross‐linking data showed that the synthetic bHLH protein, 1, was dimeric, and higher‐order aggregates of trimer and tetramer were also observed. The circular dichroism spectrum of 1 showed a high helical content, which increased upon addition of DNA containing the κE2 sequence. Gel mobility shift experiments showed that protein 1 bound sequence specifically to the κE2 sequence with a binding constant of 10−10 M2, and had an affinity for other E box sequences as well. Comparisons between the co‐crystal structure of IEB E47 with DNA and structural studies in solution showed lower helical contents in solution as would have been predicted from the crystal structure. © Munksgaard 1995.