Protein kinase A (PKA) is a prototype of multidomain signaling proteins functioning as allosteric conformational switches. Allosteric transitions have been the subject of extensive structural and dynamic investigations focusing mainly on folded domains. However, the current understanding of the allosteric role of partially unstructured linkers flanking globular domains is limited. Here, we show that a dynamic linker in the regulatory subunit (R) of PKA serves not only as a passive covalent thread, but also as an active allosteric element that controls activation of the kinase subunit (C) by tuning the inhibitory preequilibrium of a minimally populated intermediate (apo R). Apo R samples both C-binding competent (inactive) and incompetent (active) conformations within a nearly degenerate freeenergy landscape and such degeneracy maximally amplifies the response to weak (∼2RT), but conformation-selective interactions elicited by the linker. Specifically, the R linker that in the R:C complex docks in the active site of C in apo R preferentially interacts with the C-binding incompetent state of the adjacent cAMP-binding domain (CBD). These unanticipated findings imply that the formation of the intermolecular R:C inhibitory interface occurs at the expense of destabilizing the intramolecular linker/CBD interactions in R. A direct implication of this model, which was not predictable solely based on protein structure, is that the disruption of a linker/CBD salt bridge in the R:C complex unexpectedly leads to increased affinity of R for C. The linker includes therefore sites of R:C complex frustration and frustration-relieving mutations enhance the kinase inhibitory potency of R without compromising its specificity.R egulation of signaling systems often relies on multidomain proteins, which function as conformational switches controlled by allosteric effectors (1-13). The structural and dynamic changes experienced by folded domains during effector-dependent allosteric transitions have been extensively investigated (1-21). However, the current understanding of the allosteric role of partially unstructured linkers is at best scant. Although the role of covalent linkage in colocalization of protein domains is well known, it has recently been hypothesized that linkers, although generally quite flexible, have evolved to serve not simply as passive covalent threads connecting one domain to the next (i.e., "beadson-a-string" model), but also as active components of functionally relevant allosteric networks (22)(23)(24)(25). To test this hypothesis, here we have investigated the allosteric role of a critical linker in the regulatory subunit (R) of protein kinase A (PKA). This linker bridges the inhibitory site (IS) and a critical cAMP-binding domain (CBD) of R [i.e., RIα (119-244) or CBD domain A (CBD-A), ref. 26, Fig. 1A]. The R construct spanning the IS, the linker, and CBD-A [i.e., RIα (91-244) or R A in short, Fig. 1A] exhibits affinities for the catalytic subunit of PKA (C) and for cAMP as well as structures that are similar...
Focusing on the interplay of context and language, this study examined a group of high school students and their mentors' use of language during a robotics competition. This informal setting allowed us to gain insights into the mediation and manifestation of power within the group. Using critical discourse analysis of competition transcripts and interviews we found that both students and mentors felt a sense of ownership and community leading to symmetry in power amongst them. The shift in power led to greater student ownership and agency and created a space for authentic and meaningful science learning. The context of the robotics competition mediated discourse practices that were different from students' classroom experiences in that they were descriptive, relational, explanatory, and had an authentic evaluative dimension. This engaged the participants to co-construct and critique each other's knowledge claims thereby engaging in scientific practices that approximated the practices of scientists. Our study presents an argument that language and context reflexively influenced one another and reduced the imbalance of power amongst the participants thereby adding a new dimension to what has already been established about the conditions under which authentic science learning is likely to occur.
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