Spatiotemporal mapping and engineering in the dark proteome - The overview of the lab is that we investigate how protein motions associated with intrinsic disorder have specific functions. We use a range of methods including NMR, small angle scattering(SAS), and affinity measurements combined with extensive simulation of processes at atomistic or coarse-grained levels. We collaborate extensively to gain insight into a broad set of functions, while maintaining specific expertise. Our current studies are mainly focused (a) on the role of disorder of proteins lining the central transporter of the nuclear pore complex (NPC) which facilitate transport of a select group of biomolecules, mainly proteins into the nucleus, and RNA out, while keeping out others, and (b) on how to engineer triggered production of proteins in specific contexts using inteins. For the NPC, integrative structural biologists have provided a detailed picture of the framework ring structure mainly by cryo EM, and starting with that, a coarse-grained model of the central transporter was developed based on simulation and SAS, with simplified representation of the diffusing molecules and of the flexible internal proteins. The enthalpies and entropies of interactions between the passive (slow) diffusers, and facilitated diffusers with the internal proteins was obtained by experiments on isolated components and provides rates of motion for the model. Running the model with various different sizes of passive and facilitated diffusers provides a map of particles, and their rates of diffusion can be obtained and analyzed as an energy of activation required for passage through the entropic barrier of the internal proteins and interacting diffusers. Our first map provides a clear picture that discriminates the size dependence of the passive and facilitated diffusers. The weak, rapid interactions between facilitated diffusers and internal flexible proteins balances out the entropic exclusion from flexibility. This first map averages out all the different flexible proteins in the central transporter, and does not have a precise externally derived time scale, and our next steps will provide those details by production of synthetic pore models based on DNA origami, to characterize both strengths of interactions of the various flexible proteins and their interactions with diffusers, and provide an improved time scale by appropriate NMR measurements on isotopically labeled proteins. Advancing our description of NPC transport likely increases our understanding of malfunctioning NPC’s roles in cancer, ageing, viral infection, and neurodegeneration. Inteins are the proteins responsible for protein splicing, a posttranslational modification where intervening proteins (inteins) self-excise from the precursor and ligate the external sequences (exteins). This makes them excellent tools for protein engineering, using split inteins with two segments associating. One of the two segments is usually significantly disordered. We seek to expand the application of inteins to provide conditional proximity splicing by detailed analysis of the structural and dynamic features of modified split inteins. This requires engineered constructs with zero mutual association (and splicing) without trigger activation, but which turn on when triggered. We shall use the experimental and simulation tools to develop this significant application for biomedical research and potential for therapeutics e. g. triggered dual antibodies or drug conjugates at specific cell sites.
