The DNA binding domains of Cytidine Repressor (CytR) and Fructose Repressor (FruR) are paralogs with distinct conformational attributes in the absence of their binding partner, DNA. CytR is disordered in its apo- form and folds upon binding DNA, while FruR remains ordered in DNA bound- and unbound form. In this work, we extract the thermodynamic and structural determinants shaping these extreme behaviors by combining spectroscopy, mutational analysis, statistical modeling and simulations. Mimicking in vivo crowding conditions via PEG induces a continuum of order in disordered CytR, which is exploited to quantify entropic excluded volume effects and an empirical relation for estimating the expected degree of stabilization upon molecular crowding. FruR, which is marginally stable, is found to undergo a decoupled unfolding mechanism as identified by a unique and site-specific Tyr-Tyr stacking interaction, which is absent in CytR. These observations are validated by alanine-scanning mutations that destabilize FruR in a context-dependent manner providing insights on the contribution of many hydrophobic residues to the interaction network and hence the overall stability. Helix 3 which connects the DBD to the ligand binding domain is found to unfold thermodynamically earlier consistent with long time-scale molecular dynamics simulations. We thus showcase how two paralogs display diverse conformational features driven by minor but site-specific differences in their interaction network, effectively positioning one protein on the verge of order (CytR) and the other on the verge of disorder (FruR).



Publications:
1. Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein, Journal of Physical Chemistry Letters, Divya Rajendran, Shrutarshi Mitra, Hiroyuki Oikawa, Kulkarni Madhurima, Ashok Sekhar, Satoshi Takahashi, and Athi N. Naganathan, 2022, 13, 3112−3120. doi: 10.1021/acs.jpclett.2c00316
2. Flexible Target Recognition of the Intrinsically Disordered DNA-Binding Domain of CytR Monitored by Single-Molecule Fluorescence Spectroscopy, The Journal of Physical Chemistry B, Shrutarshi Mitra, Hiroyuki Oikawa, Divya Rajendran, Toshiyuki Kowada, Shin Mizukami, Athi N. Naganathan, Satoshi Takahashi, 2022, 126, 33, 6136–6147. doi: 10.1021/acs.jpcb.2c02791
3. Controlling Structure and Dimensions of a Disordered Protein via Mutations, Biochemistry, Sneha Munshi, Divya Rajendran, Samyuktha Ramesh, Sandhyaa Subramanian, Kabita Bhattacharjee, Meagha Ramana Kumar, and Athi N. Naganathan, 2020, 59, 171-174. doi:10.1021/acs.biochem.9b00678
4. Entropic Control of an Excited Folded-like Conformation in a Disordered Protein Ensemble, Journal of Molecular Biology, Sneha Munshi, Divya Rajendran and Athi N. Naganathan, 2018, 430,2688-2694. doi: 10.1016/j.jmb.2018.06.008