The Effects of Primary Sequence Perturbation on the Structure of Intrinsically Disordered Proteins




Ricard, Benjamin J.

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Intrinsically disordered proteins (IDPs) are a class of proteins that do not converge to a set of similar, energetically stable tertiary folds, but rapidly fluctuate between a wide variety of different accessible energy states. Previous work established the dependence of hydrodynamic radius (Rh) on intrinsic conformational propensity, particularly that of the polyproline-II (PPII) helix, which is in turn dependent on the primary sequence. To explore this relationship between primary sequence and structure, six IDPs having the same number and type of each amino acid derived from the intrinsically disordered N-terminus of p53, were created by substituting 11 prolines of the total 22 prolines, which has a high propensity to form the PPII helix, to glycine, which has a low propensity to form the PPII helix, in different locations; the N-terminus, the C-terminus, and the middle of the protein, as well as their retro-transposed variants, i.e. translating from the original C-terminus to the original N-terminus. Additionally, the wild-type p53, as well as its retro-transposed variant, was isolated and purified to further test direct directional dependence of structure. The propensity to form the PPII helix is measured by circular dichroism spectroscopy, and Rh is measured through size exclusion chromotography. Results showed a significant (p < 0.05) dependence of structure on directionality, as measured by differences in Rh; furthermore, circular dichroism spectra suggests that this collapse in structure is due to reduced occupancy of the PPII helix. Both directionality and location of amino acids appear to modulate the structure of IDPs indicating local charge-driven effects.



Intrinsically, Disordered, Proteins


Ricard, B. J. (2017). <i>The effects of primary sequence perturbation on the structure of intrinsically disordered proteins</i> (Unpublished thesis). Texas State University, San Marcos, Texas.


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