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Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin - PubMed

  • ️Thu Jan 01 2015

Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin

Rajkamal Balu et al. Sci Rep. 2015.

Abstract

Rec1-resilin is the first recombinant resilin-mimetic protein polymer, synthesized from exon-1 of the Drosophila melanogaster gene CG15920 that has demonstrated unusual multi-stimuli responsiveness in aqueous solution. Crosslinked hydrogels of Rec1-resilin have also displayed remarkable mechanical properties including near-perfect rubber-like elasticity. The structural basis of these extraordinary properties is not clearly understood. Here we combine a computational and experimental investigation to examine structural ensembles of Rec1-resilin in aqueous solution. The structure of Rec1-resilin in aqueous solutions is investigated experimentally using circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS). Both bench-top and synchrotron SAXS are employed to extract structural data sets of Rec1-resilin and to confirm their validity. Computational approaches have been applied to these experimental data sets in order to extract quantitative information about structural ensembles including radius of gyration, pair-distance distribution function, and the fractal dimension. The present work confirms that Rec1-resilin is an intrinsically disordered protein (IDP) that displays equilibrium structural qualities between those of a structured globular protein and a denatured protein. The ensemble optimization method (EOM) analysis reveals a single conformational population with partial compactness. This work provides new insight into the structural ensembles of Rec1-resilin in solution.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Far-UV CD spectra of Rec1-resilin measured as a function of solution pH.

Rec1-resilin displays the minimum at around 196 nm, characteristic of a random coil secondary structure.

Figure 2
Figure 2

(A) Comparison of experimental SAXS patterns of Rec1-resilin collected from both bench-top (blue) and synchrotron (black) beam lines. Inset is the corresponding Guinier approximation plot used to determine the radius of gyration (Rg) of the molecule. (B) Pair-distance distribution function, P(r), of Rec1-resilin derived from synchrotron SAXS data fit using PRIMUS program — asymmetric P(r) curve (characteristic of elongated molecule) with a maximum particle size (Dmax) estimated at ~200 angstrom.

Figure 3
Figure 3

(A) The Porod plot and (B) Dimensionless Kratky plot of Rec1-resilin derived from synchrotron SAXS data. Rec1-resilin displays the characteristics of a partially compact molecule in-solution with estimated Porod slope (−2.2 ± 0.04) between that of Gaussian chains (~2) and collapsed polymer coils (~3). The Kratky plot displays an initial monotonic increase in the lower q-region followed by a plateau with gentle negative slope in the higher q-region — the characteristics of a non-folded overall random coil secondary structural conformation.

Figure 4
Figure 4

(A) Radius of gyration (Rg) and (B) Maximum particle size (Dmax) distributions of Rec1-resilin plotted as functions of frequency (arb. unit) using the ensemble optimization method (EOM). (C) Representative ab initio 3D-model structure (one among an infinite ensemble of possible 3D-densities) of Rec1-resilin reconstructed using the GASBOR program from the distance distribution function output.

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