Biology subjects

Meador, K.

Publications and source records attributed to Meador, K..

4 recordsLinked to original sources

bioRxiv2023

Design of a symmetry-broken tetrahedral protein cage by a method of internal steric occlusion

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, e.g., so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

biochemistry↗

bioRxiv2023

A Suite of Designed Protein Cages Using Machine Learning Algorithms and Protein Fragment-Based Protocols

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, while methods for their creation remain challenging and unpredictable. In the present study, we apply new computational approaches to design a suite of new tetrahedrally symmetric, self-assembling protein cages. For the generation of docked poses, we emphasize a protein fragment-based approach, while for de novo interface design, a comparison of computational protocols highlights the power and increased experimental success achieved using the machine learning program ProteinMPNN. In relating information from docking and design, we observe that agreement between fragment-based sequence preferences and ProteinMPNN sequence inference correlates with experimental success. Additional insights for designing polar interactions are highlighted by experimentally testing larger and more polar interfaces. In all, using X-ray crystallography and cryo-EM, we report five structures for seven protein cages, with atomic resolution in the best case reaching 2.0 [A]. We also report structures of two incompletely assembled protein cages, providing unique insights into one type of assembly failure. The new set of designed cages and their structures add substantially to the body of available protein nanoparticles, and to methodologies for their creation.

biochemistry↗

bioRxiv2022

Rigidified Scaffolds for 3-Angstrom Resolution Cryo-EM of Small Therapeutic Protein Targets

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 [A] 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 [A]. 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.

biochemistry↗

bioRxiv2021

A fragment-based protein interface design algorithm for symmetric assemblies

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.

synthetic biology↗