@article {68561, title = {Time-tagged ticker tapes for intracellular recordings.}, journal = {Nature Biotechnology}, volume = {41}, year = {2023}, month = {2023 May 01}, pages = {631-9}, abstract = {

Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30-40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.

}, issn = {1546-1696}, doi = {10.1038/s41587-022-01524-7}, author = {Lin, Dingchang and Li, Xiuyuan and Moult, Eric and Park, Pojeong and Tang, Benjamin and Shen, Hao and Grimm, Jonathan B and Falco, Natalie and Jia, Bill Z and Baker, David and Lavis, Luke D and Cohen, Adam E} } @article {47711, title = {Accurate design of megadalton-scale two-component icosahedral protein complexes.}, journal = {Science (New York, N.Y.)}, volume = {353}, year = {2016}, month = {2016 Jul 22}, pages = {389-94}, abstract = {

Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

}, issn = {1095-9203}, doi = {10.1126/science.aaf8818}, author = {Bale, Jacob B and Gonen, Shane and Liu, Yuxi and Sheffler, William and Ellis, Daniel and Thomas, Chantz and Cascio, Duilio and Yeates, Todd O and Gonen, Tamir and King, Neil P and Baker, David} } @article {47562, title = {Design of a hyperstable 60-subunit protein icosahedron.}, journal = {Nature}, year = {2016}, month = {2016 Jun 15}, abstract = {

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent {\textquoteright}standard candles{\textquoteright} for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

}, issn = {1476-4687}, doi = {10.1038/nature18010}, author = {Hsia, Yang and Bale, Jacob B and Gonen, Shane and Shi, Dan and Sheffler, William and Fong, Kimberly K and Nattermann, Una and Xu, Chunfu and Huang, Po-Ssu and Ravichandran, Rashmi and Yi, Sue and Davis, Trisha N and Gonen, Tamir and King, Neil P and Baker, David} } @article {46845, title = {Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces.}, journal = {Science}, volume = {348}, year = {2015}, month = {2015 Jun 19}, pages = {1365-8}, abstract = {

We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 21 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.

}, doi = {10.1126/science.aaa9897 }, author = {Gonen, Shane and DiMaio, Frank and Gonen, Tamir and Baker, David} } @article {46923, title = {Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression.}, journal = {Protein Science}, volume = {24}, year = {2015}, month = {2015 Jul 15}, pages = {1695-701}, abstract = {

We recently reported the development of a computational method for the design of co-assembling, multi-component protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.2748}, author = {Bale, Jacob B and Park, Rachel U and Liu, Yuxi and Gonen, Shane and Gonen, Tamir and Cascio, Duilio and King, Neil P and Yeates, Todd O and Baker, David} } @article {45180, title = {Accurate design of co-assembling multi-component protein nanomaterials.}, journal = {Nature}, volume = {510}, year = {2014}, month = {2014 Jun 5}, pages = {103-8}, abstract = {

The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.

}, keywords = {Computer Simulation, Crystallography, X-Ray, Drug Design, Models, Molecular, Nanostructures, Protein Subunits, Proteins}, issn = {1476-4687}, doi = {10.1038/nature13404}, author = {King, Neil P and Bale, Jacob B and Sheffler, William and McNamara, Dan E and Gonen, Shane and Gonen, Tamir and Yeates, Todd O and Baker, David} } @article {45637, title = {High thermodynamic stability of parametrically designed helical bundles.}, journal = {Science}, volume = {346}, year = {2014}, month = {2014 Oct 24}, pages = {481-5}, abstract = {

We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold \> 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.

}, issn = {1095-9203}, doi = {10.1126/science.1257481}, author = {Huang, Po-Ssu and Oberdorfer, Gustav and Xu, Chunfu and Pei, Xue Y and Nannenga, Brent L and Rogers, Joseph M and DiMaio, Frank and Gonen, Tamir and Luisi, Ben and Baker, David} } @article {4920, title = {Computational design of self-assembling protein nanomaterials with atomic level accuracy.}, journal = {Science}, volume = {336}, year = {2012}, month = {2012 Jun 1}, pages = {1171-4}, abstract = {

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

}, issn = {1095-9203}, doi = {10.1126/science.1219364}, author = {King, Neil P and Sheffler, William and Sawaya, Michael R and Vollmar, Breanna S and Sumida, John P and Andr{\'e}, Ingemar and Gonen, Tamir and Yeates, Todd O and Baker, David} } @article {3635, title = {Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system.}, journal = {mBio}, volume = {1}, year = {2010}, month = {2010}, abstract = {

The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.

}, keywords = {Bacterial Proteins, Membrane Transport Proteins, Models, Molecular, Protein Binding, Protein Multimerization, Salmonella typhimurium}, issn = {2150-7511}, doi = {10.1128/mBio.00158-10}, author = {Sanowar, Sarah and Singh, Pragya and Pfuetzner, Richard A and Andr{\'e}, Ingemar and Zheng, Hongjin and Spreter, Thomas and Strynadka, Natalie C J and Gonen, Tamir and Baker, David and Goodlett, David R and Miller, Samuel I} }