A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.
Theoretical and experimental advances in protein engineering have led to the creation of precisely defined, novel protein assemblies of great size and complexity, with diverse applications. One powerful approach involves designing a new attachment or binding interface between two simpler symmetric oligomeric protein components. The required methods of design, which present both similarities and key differences compared to problems in protein docking, remain challenging and are not yet routine. With the aim of more fully enabling this emerging area of protein material engineering, we developed a computer program, nanohedra, to introduce two key advances. First, we encoded in the program the construction rules (i.e. the search space parameters) that underlie all possible symmetric material constructions. Second, we developed algorithms for rapidly identifying favorable docking/interface arrangements based on tabulations of empirical patterns of known protein fragment-pair associations. As a result, the candidate poses that nanohedra generates for subsequent amino acid interface design appear highly native-like (at the protein backbone level), while simultaneously conforming to the exacting requirements for symmetry-based assembly. A retrospective computational analysis of successful vs failed experimental studies supports the expectation that this should improve the success rate for this challenging area of protein engineering.
Numerous technical advances have made cryo-EM an attractive method for atomic structure determination. Cryo-EM is ideally suited for large macromolecular structures, while problems of low-signal-to-noise prevent routine structure determination of proteins smaller than about 50 kDa. This size limitation excludes large numbers of important cellular proteins from structural characterization by this powerful technique, including many cell-signaling proteins of high therapeutic interest. In the present work, we use molecular engineering techniques to rigidify an imaging scaffold, based on a designed protein cage, to the point where 3 Å resolution can be achieved, even for very small proteins. After optimizing the design of the rigidified scaffold on test proteins, we apply this imaging system to the key oncogenic signaling protein KRas, which represents an outstanding challenge in the area of structure-based drug design. Despite its 19 kDa size, we show that the structure of KRas, in multiple mutant forms, and bound to its GDP ligand, can be readily interpreted at a resolution slightly better than 3.0 Å. This advance further expands the capability of cryo-EM to become an essentially universal method for protein structure determination, including for applications to small therapeutic protein targets.
Theoretical and experimental advances in protein engineering have led to the creation of precisely defined, novel protein assemblies of great size and complexity, with diverse applications. One powerful approach involves designing a new attachment or binding interface between two simpler symmetric oligomeric protein components. The required methods of design, which present both similarities and key differences compared to problems in protein docking, remain challenging, and are not yet routine. With the aim of more fully enabling this emerging area of protein material engineering, we developed a computer program, Nanohedra, to introduce two key advances. First, we encoded in the program the construction rules (i.e. the search space parameters) that underlie all possible symmetric material constructions. Second, we developed algorithms for rapidly identifying favorable docking/interface arrangements based on tabulations of empirical patterns of known protein fragment-pair associations. As a result, the candidate poses that Nanohedra generates for subsequent amino acid interface design appear highly native-like (at the protein backbone level), while simultaneously conforming to the exacting requirements for symmetry-based assembly. A retrospective computational analysis of successful vs failed experimental studies supports the expectation that this should improve the success rate for this challenging area of protein engineering.
Numerous technical advances have made cryo-EM an attractive method for atomic structure determination. Cryo-EM is ideally suited for very large structures; symmetrical structures like viruses are especially amenable. However, problems of low-signal-to-noise in imaging small proteins makes it practically impossible to determine structures smaller than about 50 kDa, leaving a great many cellular proteins and enzymes (and nucleic acid molecules) outside the reach of this important structural technique. We have developed symmetric protein imaging scaffolds to display and solve the structure of small proteins. In earlier work (Liu Y, Huynh DT, Yeates TO. A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. Nat Commun. 2019), we broke through this barrier by engineering novel scaffolds with sufficient rigidity and modularity to achieve resolution useful for interpreting atomic structure, reaching 3.8 Å resolution for a 26 kDa protein. To overcome the challenges of flexibility between the protein target and the imaging scaffold and further improve resolution, we have developed new computational tools to model (and then limit) range-of-motion by designing additional interfaces to rigidify our imaging scaffolds. With the new rigidified scaffolds, we can solve structures of small proteins at near-atomic resolution, reaching 3 Å resolution for sub-30 kDa protein targets. Current examples will be presented.
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