Controlling Structure and Dimensions of a Disordered Protein via Mutations

Munshi, Sneha; Rajendran, Divya; Ramesh, Samyuktha; Subramanian, Sandhyaa; Bhattacharjee, Kabita; Kumar, Meagha Ramana; Naganathan, Athi N.

doi:10.1021/acs.biochem.9b00678

Cited by 5 publications

(6 citation statements)

References 35 publications

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“…Despite our nascent understanding of the mechanisms that encode disordered ensembles within primary sequences, we are beginning to see early evidence of the potential "tunability" of the structural propensities of IDRs through point mutations. For example, Munshi et al 87 modulated the dimensions of CytR DNAbinding domain, a high sequence-complexity IDP, through the introduction of rationally positioned point mutations. These mutations resulted in a maximal reduction in the Stokes radius of ∼3.5 Å of the disordered CytR ensemble, corresponding to 40% reduction in occupied volume compared to the mutant with the most extended set of conformers.…”

Section: ■ Mechanisms Of Collapse and Aggregationmentioning

confidence: 99%

Physical Chemistry of the Protein Backbone: Enabling the Mechanisms of Intrinsic Protein Disorder

Drake

Pettitt

2020

J. Phys. Chem. B

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Over the last two decades it has become clear that well-defined structure is not a requisite for proteins to properly function. Rather, spectra of functionally competent, structurally disordered states have been uncovered requiring canonical paradigms in molecular biology to be revisited or reimagined. It is enticing and oftentimes practical to divide the proteome into structured and unstructured, or disordered, proteins. While function, composition, and structural properties largely differ, these two classes of protein are built upon the same scaffold, namely, the protein backbone. The versatile physicochemical properties of the protein backbone must accommodate structural disorder, order, and transitions between these states. In this review, we survey these properties through the conceptual lenses of solubility and conformational populations and in the context of protein-disorder mediated phenomena (e.g., phase separation, order–disorder transitions, allostery). Particular attention is paid to the results of computational studies, which, through thermodynamic decomposition and dissection of molecular interactions, can provide valuable mechanistic insight and testable hypotheses to guide further solution experiments. Lastly, we discuss changes in the dynamics of side chains and order–disorder transitions of the protein backbone as two modes or realizations of “entropic reservoirs” capable of tuning coupled thermodynamic processes.

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Section: ■ Mechanisms Of Collapse and Aggregationmentioning

confidence: 99%

Physical Chemistry of the Protein Backbone: Enabling the Mechanisms of Intrinsic Protein Disorder

Drake

Pettitt

2020

J. Phys. Chem. B

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“…The disordered DNA-binding domain (DBD) from Escherichia coli CytR (cytidine repressor) can serve as a unique model system for exploring this approach, given that the native ensemble is at the threshold of order. Specifically, mutations , (hydrophobic or charged residue substitutions), small changes in solvent conditions , (salts, independent of charge density or identity, and TMAO), and the presence of DNA , (specific or nonspecific binding sites) can drive the system toward the folded conformation (Figure ). The resulting unfolding curves and thermodynamics of (de)stabilization are also amenable to detailed characterization via the statistical mechanical Wako–Saitô–Muñoz–Eaton (WSME) model, ,, enabling an ensemble-based description while also accounting for diverse intramolecular energetics.…”

mentioning

confidence: 99%

Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein

Rajendran

Mitra

Oikawa

et al. 2022

J. Phys. Chem. Lett.

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We investigate the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol (PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-molecule Forster resonance energy transfer measurements, we demonstrate that disordered CytR populates a well-folded minor conformation in its native ensemble, while the unfolded ensemble collapses and folds with an increase in crowder density independent of the crowder size. Employing a statistical−mechanical model, the effective reduction in the accessible conformational space of a residue in the unfolded state is estimated to be 10% at 300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG−temperature phase diagram thus constructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol −1 , driving the equilibrium toward folded conformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.

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“…Analysis of the structure and dynamics of the CytR DBD allows us to further hypothesize about the link between protein flexibility and DNA recognition. The intrinsic disorder of the free CytR DBD has been confirmed and compared to LacR [7,[13][14][15][16][17][18]. Our characterization of the nonspecifically bound state classify CytR as forming a fuzzy complex with DNA where the bound protein is only partially structured [19][20][21].…”

Section: Discussionmentioning

confidence: 60%

“…Thus, the CytR DBD can be characterized as an intrinsically disordered protein (IDP). Since our CytR DBD structure was published, it has been used as a paradigm in understanding IDPs in comparison to other folded LacR DBDs [7,[13][14][15][16][17][18]].…”

Section: Introductionmentioning

confidence: 99%

Dynamic Consequences of Specificity within the Cytidine Repressor DNA-Binding Domain

Moody¹,

Soto²,

Tretyachenko-Ladokhina³

et al. 2021

Preprint

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The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that regulates nine operons with distinct spacing and orientations of recognition sites. Understanding the structural features of the CytR DNA-binding domain (DBD) when bound to DNA is critical to understanding differential mechanisms of gene regulation. We previously reported the structure of the CytR DBD monomer bound specifically to half-site DNA and found that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif, similar to other proteins that interact with DNA [Moody, et al (2011), Biochemistry 50:6622-32]. We also studied the free state of the monomer and found that since NMR spectra show it populates up to four distinct conformations, the free state exists as an intrinsically disordered protein (IDP). Here, we present further analysis of the DBD structure and dynamics in the context of full-site operator or nonspecific DNA. DBDs bound to full-site DNA show one set of NMR signals, consistent with fast exchange between the two binding sites. When bound to full-length DNA, we observed only slight changes in structure compared to the monomer structure and no folding of the hinge helix. Notably, the CytR DBD behaves quite differently when bound to nonspecific DNA compared to LacR. A dearth of NOEs and complete lack of protection from hydrogen exchange are consistent with the protein populating a flexible, molten state when associated with DNA nonspecifically, similar to fuzzy complexes. The CytR DBD structure is significantly more stable when bound specifically to the udp half-site substrate. For CytR, the transition from nonspecific association to specific recognition results in substantial changes in protein mobility that are coupled to structural rearrangements. These effects are more pronounced in the CytR DBD compared to other LacR family members.

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Controlling Structure and Dimensions of a Disordered Protein via Mutations

Cited by 5 publications

References 35 publications

Physical Chemistry of the Protein Backbone: Enabling the Mechanisms of Intrinsic Protein Disorder

Physical Chemistry of the Protein Backbone: Enabling the Mechanisms of Intrinsic Protein Disorder

Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein

Dynamic Consequences of Specificity within the Cytidine Repressor DNA-Binding Domain

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