ORCID

https://orcid.org/0000-0002-2822-540X

Date of Award

Spring 5-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Physics

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Many 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 Genetic Algorithm for Design of Intrinsic secondary Structure (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.There is a significant collection of proteins with repeating blocks of oppositely charged residues where the consensus sequence is a block of four Glu residues followed by a block of four Lys or Arg residues, (Glu4(Lys/Arg)4)n. These proteins have been experimentally shown to form long single alpha helices (SAHs) under biologically relevant conditions. However, these results are confounding to disorder predictors and to certain atomistic simulations in that both predict these sequences to be strongly disordered. The current working hypothesis is that SAHs are stabilized by i:i+4 salt bridges between opposite charges in consecutive helical turns. We test the merits of this hypothesis to understand the sequence-encoded preference for SAHs and the logic behind the failure of certain atomistic simulations in anticipating the preference for stable SAHs.In simulations with fixed charges the favorable free energy of solvation of charged residues and the associated loss of sidechain entropy hinders the formation of SAHs. We proposed that alterations to charge states induced by sequence context might play an important role in stabilizing SAHs. We tested this hypothesis using a (Glu4Lys4)n repeat protein and a simulation strategy that permits the substitution of charged residues with neutralized protonated or deprotonated variants of Glu / Lys. Our results predict that stable SAH structures derive from the neutralization of approximately half the Glu residues. These findings explain experimental observations and also provide a coherent rationale for the failure of simulations based on fixed charge models. Large-scale sequence analysis reveals that naturally occurring sequences often include "defects" in charge patterns such as Gln or Ala substitutions. This sequence-encoded incorporation of uncharged residues combined with neutralization of charged residues might tilt the balance toward alpha helical conformations.Micron-sized, non-membrane bound cellular bodies can form as the result of collective interactions between modules of distinct multidomain proteins. Li et al. have examined the phase diagrams that result for polymers of SH3 domains and proline-rich modules (PRMs) while varying the number of interacting domains. It is noteworthy that flexible, intrinsically disordered linkers connect the interacting units within each polymer. Conventional wisdom holds that linkers play a passive role in determining the phase behavior of multidomain proteins that undergo phase separations. Here, we ask if this view is accurate. The motivation for our work comes from recent studies that have uncovered a rich diversity of composition-to-conformation and sequence-to-conformation relationships for intrinsically disordered proteins. The central finding is that disordered regions of proteins have distinct sequence-encoded conformational preferences. Accordingly, we reasoned that the conformational properties of linkers might be a contributing factor, in addition to polyvalency, to the phase behavior of multidomain proteins.We have developed and deployed a three-dimensional lattice model to arrive at a predictive framework to query the effects of linkers on the phase diagrams of polyvalent systems. We find that the critical concentration for phase transition can be influenced by the conformational properties of linkers. Specifically, our results show that linkers modulate the cooperative binding between domains of polymers that are already bound together. Depending on their conformational properties, linkers can also block access to the binding domains via excluded volume effects. Additionally, we find that the properties of the linkers can lead to controls over the mixing of proteins in these bodies. Specifically, we find that there are large ranges of parameters for three protein systems where the bodies isolate specific proteins to different regions of the bodies instead of uniformly mixing them. This result is validated by recent findings of organization inside some observed bodies.

Language

English (en)

Chair and Committee

Rohit V. Pappu & Ralf Wessel

Committee Members

Greg Bowman, Anders E. Carlsson, Zohar Nussinov,

Comments

Permanent URL: https://doi.org/10.7936/K7DF6PNH

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