Cells collect and process information about the external world through elaborate networks of proteins and small molecules that we call the signal transduction machinery. The functional capability of the transduction machinery is often prodigious;weak input signals can be selectively amplified and filtered for optimal sensing of physiological stimuli, and layers of feedback control provide for complex regulatory characteristics and protection against output saturation. For example, sensory neurons of the eye can generate reliable electrical responses to the absorption of single photons under dark adapted conditions, but can also adjust the gain to continue operating in bright daylight conditions without response saturation. The overall goal of our research program is to understand how signaling systems provide for such functional characteristics, and how disease processes arise from defects in the underlying mechanisms. In this application, we propose to mechanistically understand a specific macromolecular complex that is required for visual function in Drosophila. The complex is assembled by a multi-PDZ domain protein called InaD, and our recent work shows that rather than acting as a passive scaffold, this protein is a dynamic machine that undergoes light-dependent conformational changes at the time scale of visual signaling. In the course of the experiments proposed here, we will (1) understand the exact physiological processes controlled by InaD dynamics, (2) define how light-signaling is coupled to the conformational switching of InaD, (3) define how the conformational change regulates the visual output, and (4) carry out studies to see if InaD organizes spatial gradients of signaling molecules. InaD has already served as a classic model system for understanding macromolecular organization of signaling systems. We expect that this work will expose new mechanisms of InaD function, and will help resolve current models for scaffolding in general. Scaffolding is thought to enhance the efficiency and specificity of signaling events in many cells, and defects in scaffolding have been associated with diverse disease processes, including cancer, chronic inflammaton, and developmental abnormalities. By showing how scaffolding proteins can alsoactively shape signaling events through dynamical conformational change, this work should help extend our understanding of the normal and pathological states of cellular signlaing systems.
| Rivoire, Olivier; Reynolds, Kimberly A; Ranganathan, Rama (2016) Evolution-Based Functional Decomposition of Proteins. PLoS Comput Biol 12:e1004817 |
| Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H et al. (2016) A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing. J Vis Exp : |
| Raman, Arjun S; White, K Ian; Ranganathan, Rama (2016) Origins of Allostery and Evolvability in Proteins: A Case Study. Cell 166:468-480 |
| Stiffler, Michael A; Hekstra, Doeke R; Ranganathan, Rama (2015) Evolvability as a function of purifying selection in TEM-1 ?-lactamase. Cell 160:882-892 |
| Reynolds, Kimberly A; Russ, William P; Socolich, Michael et al. (2013) Evolution-based design of proteins. Methods Enzymol 523:213-35 |
| McLaughlin Jr, Richard N; Poelwijk, Frank J; Raman, Arjun et al. (2012) The spatial architecture of protein function and adaptation. Nature 491:138-42 |
| Reynolds, Kimberly A; McLaughlin, Richard N; Ranganathan, Rama (2011) Hot spots for allosteric regulation on protein surfaces. Cell 147:1564-75 |
| Halabi, Najeeb; Rivoire, Olivier; Leibler, Stanislas et al. (2009) Protein sectors: evolutionary units of three-dimensional structure. Cell 138:774-86 |
