Browsing by Subject "Macromolecular Substances"
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Item Recruitment of Enzyme Cascade to Phase-Separated Biomolecular Condensates Accelerates Reactions via Concentration-Dependent and Concentration-Independent Mechanisms(August 2021) Peeples, William Benjamin; De Martino, George; Phillips, Margaret A.; Kohler, Jennifer J.; Rosen, Michael K.Biomolecular condensates are ubiquitous throughout biology, but their functions remain largely poorly understood. Biomolecular condensates concentrate biomolecules relative to the surrounding medium. For biomolecular condensates that concentrate enzymes and their substrates, classic enzyme kinetics predicts an acceleration of the reaction rate within the condensates, but the effect of condensates on enzymatic activity both within and outside condensates has not been widely investigated. In order to understand these effects in more detail, we developed an in vitro model system consisting of multivalent protein scaffolds and a minimal enzyme system-the SUMOylation cascade. By inducibly recruiting various combinations of components of the SUMOylation cascade to condensates, we are able to uncouple the contributions of individual components and phases to enzymatic activity. We find that the reaction is accelerated when all SUMOylation components are recruited to condensates, and this acceleration requires recruitment of both enzyme and substrate. This is despite condensates representing only 1 % of total solution volume. This enhancement is limited to substrates whose KM is well above total substrate concentration. This selective enhancement is further demonstrated with simple modeling to show that substrate concentration relative to KM is a key factor in understanding the degree to which different substrates are likely to be influenced through condensate recruitment. Recruitment accelerates not only the reaction within the condensate but also the reaction outside the condensates. To understand what fraction of this increased activity within condensates is attributable to increased concentration of enzyme and substrate, we measured activity at identical concentrations of enzyme and substrate but lacking the scaffolds. We find that condensate activity exceeds the concentration-matched reaction, suggesting there is concentration-independent activity enhancement. Further investigation found that this excess enhancement is likely due to a scaffold-induced reduction in apparent KM. These results suggest that condensates can accelerate enzymatic activity through multiple mechanisms, including concentration and molecular organization of enzyme and substrate. Condensates selectively accelerate substrates whose total concentration is low relative to KM. Together these effects demonstrate the capacity of condensates to impart activity enhancement, specificity, and potentially sequestration through regulated enzyme and substrate recruitment.Item The Role of the Scaffolding Protein INAD in Localization of Signaling Complexes to the Rhabdomeres of Drosophil Photoreceptors(2004-12-15) Hahn, Adrienne R.; Ranganathan, RamaOrganization of proteins into macromolecular complexes is one way cells maximize the speed, specificity, and efficiency of signal transduction. In fruit fly photoreceptors InaD, a scaffolding protein containing 5 PDZ domains, organizes proteins involved in the visual signaling pathway into complexes within a microvillar stack of membranes known as the rhabdomere. Light activation of rhodopsin activates a signaling cascade via a Gq-coupled reaction that quickly opens Ca++-selective TRP channels. Subsequent Ca++ influx activates eye protein kinase C (ePKC), and calmodulin, which in turn modulate the activity of other visual proteins. Mutants in which the InaD / TRP association has been disrupted (inaD215) phenocopy the delayed inactivation of mutants lacking ePKC, suggesting that one of the functions of InaD includes localizing ePKC to its downstream targets such as the TRP channel. There are currently two different models for scaffolding proteins: the "beads on a string" or "tethering" model where the order of the binding domains and their respective binding partners is unimportant, and the "specific quaternary structure" model where the specific stereochemical orientation of the domains is vital for proper signaling. The latter model also allows for allosteric regulation of binding. We assess the "beads on a string" vs. the "specific quaternary structure" model for InaD, a scaffolding protein found in the photoreceptor cells of fruit flies, by analyzing the characteristics of the light response in flies expressing two InaD constructs where the order of the PDZ domains has been shuffled. Based on biochemical and electrophysiological data on these mutants, we conclude that the "specific quaternary structure" model applies best to InaD. In addition, we investigate whether the inaD215 phenotype is due to displacement of ePKC from microdomains of calcium initiated by TRP channels, by calcium imaging of photoreceptors expressing visual proteins tagged with CaMgaroo, a Ca++-sensitive derivative of yellow fluorescent protein. After conducting these experiments, we conclude that this is not the case. Rather, the inaD215 phenotype is most likely due to the inability of ePKC to phosphorylate TRP and and attenuate its activity. These results also support the "specific quaternary structure" model for InaD.