Eswar Reddy Maddi, Sathees C Raghavan, Ramanathan Natesh
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Hypomorphic mutations in human DNA ligase IV lead to compromised DNA binding efficiency, hydrophobicity and thermal stability.
Studies have shown that Lig4 syndrome mutations in DNA ligase IV (LigIV) are compromised in its function with residual level of double strand break ligation activity in vivo. It was speculated that Lig4 syndrome mutations adversely affect protein folding and stability. Though there are crystal structures of LigIV, there are no reports of crystal structures of Lig4 syndrome mutants and their biophysical characterization to date. Here, we have examined the conformational states, thermal stability, hydrophobicity and DNA binding efficiency of human DNA LigIV wild type and its hypomorphic mutants by far-UV circular dichroism, tyrosine and tryptophan fluorescence, and 1-anilino-8-naphthalene-sulfonate binding, dynamic light scattering, size exclusion chromatography, multi-angle light scattering and electrophoretic mobility shift assay. We show here that LigIV hypomorphic mutants have reduced DNA-binding efficiency, a shift in secondary structure content from the helical to random coil, marginal reduction in their thermal stability and increased hydrophobicity as compared to the wild-type LigIV.
期刊介绍:
Protein Engineering, Design and Selection (PEDS) publishes high-quality research papers and review articles relevant to the engineering, design and selection of proteins for use in biotechnology and therapy, and for understanding the fundamental link between protein sequence, structure, dynamics, function, and evolution.