Abstract
Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements.
Highlights
Many macromolecular complexes involve proteins or regions that are intrinsically disordered in their unbound forms (Wright and Dyson, 1999, 2009, Babu et al, 2012, van der Lee et al, 2014, Wright and Dyson, 2015)
The amino acid sequences of intrinsically disordered proteins (IDPs) encode an intrinsic preference for conformational heterogeneity, which means that they do not fold into specific threedimensional structures as autonomous units (Dunker et al, 2002)
In its PUMA bound to Residue Specific Helicity Probability unbound state, PUMA adopts a heterogeneous ensemble of partially helical conformations (Fig. 2)
Summary
Many macromolecular complexes involve proteins or regions that are intrinsically disordered in their unbound forms (Wright and Dyson, 1999, 2009, Babu et al, 2012, van der Lee et al, 2014, Wright and Dyson, 2015). A particular value for the dissociation constant (KD) can accommodate a range of mechanisms for coupled folding and binding (Kiefhaber et al, 2012) This feature is highlighted in kinetics experiments that have measured the rates of association of the intrinsically disordered BH3-PUMA (referred to hereafter as PUMA) peptide to the pre-folded Mcl-1 (Rogers et al, 2013, 2014a,b) and other systems (Dogan et al, 2015). An intriguing hypothesis is that the amino acid composition of an IDP is the main determinant of kon whereas the degree of intrinsic helicity regulates koff leading to kinetic control of cellular programs such as apoptosis To test this hypothesis, one needs a systematic titration of the effects of intrinsic helicity on the mechanisms of coupled folding and binding. Mutagenesis experiments inevitably convolve changes to amino acid composition and intrinsic helicities, as is the case with standard, proline-, glycine- or alanine-scanning approaches. Quantitative comparisons show that computationally derived mean helicities are in agreement with those derived from experiment
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