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bioRxiv · 10.1101/2024.07.05.602234

Structural Characterization of DNA Binding Domain of Essential Mammalian Protein TTF 1

Singh, G.·Bhopale, A. J.·Khatri, S.·Prakash, P.·Kumar, R.·Singh, S. M.·Singh, S. K.
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Abstract

Transcription termination factor 1 (TTF1), being multifunctional in nature, is involved in a wide range of critical processes that make it essential for survival of the mammalian cells. TTF1 protein comprises three functional domains: the N-terminal (regulatory/inhibitory) domain, trans-activation domain, and C-terminal domain. The Myb domain is responsible for DNA-binding function of this protein and spans 550 to 732 amino acids (183 residues long). Despite the essential role of TTF1 in multiple cellular processes, there is no physical structure available to date. Purification of the functional full-length protein has been unsuccessful so far. Hence, we moved forward towards characterizing the Myb domain of this essential protein. We first constructed a three-dimensional model of the Myb domain using Robetta server and determined its stability through MD simulation in an explicit solvent. To validate the model, upon codon optimization we cloned this domain into a bacterial expression vector. The protein was then purified to homogeneity and its DNA-binding activity was checked by electro-mobility shift assay. We then proceeded to CD spectroscopy and Raman spectroscopy for secondary structure characterization. The results validated the computational model, concluding that this domain is predominantly helical in nature. The confidence built by this study now pushes us to move ahead in order to solve the atomic structure of this critical domain by crystallography or NMR spectroscopy, which in turn will decipher the exact mechanism by which this essential protein engages DNA to cater to various functions.

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Singh, G., Bhopale, A. J., Khatri, S., Prakash, P., Kumar, R., Singh, S. M., Singh, S. K.. 2024-07-06. Structural Characterization of DNA Binding Domain of Essential Mammalian Protein TTF 1. https://doi.org/10.1101/2024.07.05.602234

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