Synthesis of the basic-helix-loop-helix region of the immunoglobulin enhancer binding protein E47 and evaluation of its structural and DNA binding properties.
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 kappa E2 sequence. Gel mobility shift experiments showed that protein 1 bound sequence specifically to the kappa E2 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.
Bishop
P
P
Department of Chemistry, Purdue University, West Lafayette, Indiana, USA.
Jones
C
C
Ghosh
I
I
Chmielewski
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D015342:DNA Probes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D015342:DNA Probes; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors