Engineering Order and Cooperativity in a Disordered Protein

Munshi, Sneha; Subramanian, Sandhyaa; Ramesh, Samyuktha; Golla, Hemashree; Divakar, K.; Madhurima, Kulkarni; Campos, Luis Alberto; Sekhar, Ashok; Naganathan, Athi N.

doi:10.1021/acs.biochem.9b00182

Cited by 10 publications

(36 citation statements)

References 64 publications

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“… 18 , 30 In fact, even the folded-like DM displays broad, weakly cooperative, and probe-dependent unfolding. 20 Taken together, our observations suggest that the apparent Stokes radius represents an effective ensemble average of numerous partially structured states.…”

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confidence: 56%

“…(B) Size-exclusion chromatograms of the mutants in panel A at 298 K, 150 mM ionic strength ammonium acetate buffer at pH 8.0. 20 A high ionic strength is required to eliminate nonspecific interactions of proteins with the column matrix. The reported dimensions are thus lower estimates as CytR reduces its dimensions with salt.…”

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confidence: 99%

“… 17 The conformational ensemble of CytR is sensitive to temperature, 18 salt, 19 and DNA, 17 , 19 all of which arise from a combination of destabilizing electrostatics ( Figure 1B ) and weak packing in the folded conformation. 20 In this work, we control the dimensions of the disordered CytR via a combination of folded structure and sequence-alignment-based expectations, and rational engineering. Though we observe nonintuitive effects, a likely manifestation of non-native interactions within the disordered ensemble, we find a strong correlation between secondary structure and the apparent Stokes radius ( R s ), a first such observation in IDPs.…”

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confidence: 99%

“…True to this, the thermal dependence of the P33G mutant’s CD signal is flat (blue in Figure 2A ) unlike the weak structural transitions observed in the WT or the P33A mutant (red and green in Figure 2A , respectively). In addition to the secondary structure content, the mean dimensions as measured by R s (in a calibrated size-exclusion chromatography column 20 ), shows a trend where P33G is more expanded than the WT ( Figure 2B ). Another interesting position is residue K35 located in the electrostatically frustrated binding site of the folded conformation with a number of positive charges around it ( Figure 1B ).…”

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confidence: 99%

“…Similarly, we have shown recently that the double mutant A29V/A48M (DM; that introduces two large hydrophobic substitutions in the protein core) promotes collapse of the disordered ensemble into a compact folded ensemble while simultaneously increasing the secondary-structure content (mutations identified via sequence comparisons; magenta in Figure 2 and Supporting Figure S1 ). 20 Building on the folded-like conformation of the DM, we engineered two other mutants, Quad (A29V/A48M/R43A/K46A; black in Figure 2 ) and Pent (A29V/A48M/R43A/K46A/R28A; gray in Figure 2 ), that further eliminate the residual electrostatic frustration. These two mutants again promote structure and compaction while differentially affecting the secondary structure content.…”

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confidence: 99%

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Controlling Structure and Dimensions of a Disordered Protein via Mutations

et al. 2019

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The dimensions of intrinsically disordered proteins (IDPs) are sensitive to small energetic-entropic differences between intramolecular and protein–solvent interactions. This is commonly observed on modulating solvent composition and temperature. However, the inherently heterogeneous conformational landscape of IDPs is also expected to be influenced by mutations that can (de)stabilize pockets of local and even global structure, native and non-native, and hence the average dimensions. Here, we show experimental evidence for the remarkably tunable landscape of IDPs by employing the DNA-binding domain of CytR, a high-sequence-complexity IDP, as a model system. CytR exhibits a range of structure and compactness upon introducing specific mutations that modulate microscopic terms, including main-chain entropy, hydrophobicity, and electrostatics. The degree of secondary structure, as monitored by far-UV circular dichroism (CD), is strongly correlated to average ensemble dimensions for 14 different mutants of CytR and is consistent with the Uversky–Fink relation. Our experiments highlight how average ensemble dimensions can be controlled via mutations even in the disordered regime, the prevalence of non-native interactions and provide testable controls for molecular simulations.

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Controlling Structure and Dimensions of a Disordered Protein via Mutations

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Simple mechanisms for the evolution of protein complexity

Pillai

Hochberg

2022

Protein Science

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Proteins are tiny models of biological complexity: specific interactions among their many amino acids cause proteins to fold into elaborate structures, assemble with other proteins into higher‐order complexes, and change their functions and structures upon binding other molecules. These complex features are classically thought to evolve via long and gradual trajectories driven by persistent natural selection. But a growing body of evidence from biochemistry, protein engineering, and molecular evolution shows that naturally occurring proteins often exist at or near the genetic edge of multimerization, allostery, and even new folds, so just one or a few mutations can trigger acquisition of these properties. These sudden transitions can occur because many of the physical properties that underlie these features are present in simpler proteins as fortuitous by‐products of their architecture. Moreover, complex features of proteins can be encoded by huge arrays of sequences, so they are accessible from many different starting points via many possible paths. Because the bridges to these features are both short and numerous, random chance can join selection as a key factor in explaining the evolution of molecular complexity.

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Slow Folding of a Helical Protein: Large Barriers, Strong Internal Friction, or a Shallow, Bumpy Landscape?

et al. 2020

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The rate at which a protein molecule folds is determined by opposing energetic and entropic contributions to the free energy that shape the folding landscape. Delineating the extent to which they impact the diffusional barrier-crossing events, including the magnitude of internal friction and barrier height, has largely been a challenging task. In this work, we extract the underlying thermodynamic and dynamic contributions to the folding rate of an unusually slow-folding helical DNA-binding domain, PurR, which shares the characteristics of ultrafast downhill-folding proteins but nonetheless appears to exhibit an apparent two-state equilibrium. We combine equilibrium spectroscopy, temperature-viscosity-dependent kinetics, statistical mechanical modeling, and coarse-grained simulations to show that the conformational behavior of PurR is highly heterogeneous characterized by a large spread in melting temperatures, marginal thermodynamic barriers, and populated partially structured states. PurR appears to be at the threshold of disorder arising from frustrated electrostatics and weak packing that in turn slows down folding due to a shallow, bumpy landscape and not due to large thermodynamic barriers or strong internal friction. Our work highlights how a strong temperature dependence on the pre-exponential could signal a shallow landscape and not necessarily a slow-folding diffusion coefficient, thus determining the folding timescales of even millisecond folding proteins and hints at possible structural origins for the shallow landscape.

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Engineering Order and Cooperativity in a Disordered Protein

Cited by 10 publications

References 64 publications

Controlling Structure and Dimensions of a Disordered Protein via Mutations

Controlling Structure and Dimensions of a Disordered Protein via Mutations

Simple mechanisms for the evolution of protein complexity

Slow Folding of a Helical Protein: Large Barriers, Strong Internal Friction, or a Shallow, Bumpy Landscape?

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