title: Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors.
abstract: Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.
authors.affiliation: Biomolecular Science and Engineering Program.
authors.forename: Anna J
authors.initials: AJ
authors.lastname: Simon
authors.affiliation: Laboratory of Biosensors and Nanomachines, Département de Chimie, Université de Montréal, Montreal, QC, Canada H3T 1J4; and.
authors.forename: Alexis
authors.initials: A
authors.lastname: Vallée-Bélisle
authors.affiliation: Dipartimento di Scienze e Tecnologie Chimiche, University of Rome, Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Ricci
authors.affiliation: Biomolecular Science and Engineering Program, Department of Chemistry and Biochemistry, and Center for Bioengineering, University of California, Santa Barbara, CA 93106; kwp@chem.ucsb.edu.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Plaxco
mesh_terms: D000495:Allosteric Site; D015374:Biosensing Techniques; D001709:Biotechnology / Q000379:methods*; D003042:Cocaine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004317:Doxorubicin / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D008024:Ligands; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D016337:RNA, Catalytic / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D005452:Fluoresceins; D008024:Ligands; D016337:RNA, Catalytic; C024098:6-carboxyfluorescein; D004317:Doxorubicin; D004247:DNA; D003042:Cocaine; D010100:Oxygen
keywords: biosensors; intrinsically disordered proteins; ribozymes; synthetic biology; ultrasensitivity