De novo design of protein logic gates

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions

De novo design of knotted tandem repeat proteins.

Acknowledgements: We thank Meredith Steward for assistance with biochemical experiments. Support for this work was provided by the Fred Hutchinson Cancer Center and by the National Institute for General Medical Sciences (NIGMS) for both BLS (R01 GM139752) and PB (R35 GM141457). L.F.M. was supported by a Human Frontier Science Program Cross Disciplinary Fellowship (LT000395/2020-C) and an EMBO Non-Stipendiary Fellowship (ALTF 1047-2019). Crystallographic data collection was conducted at the Advanced Light Source (ALS) at the Berkeley Center for Structural Biology which is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231. The Pilatus detector at Beamline 5.0.1 (where the data was collected) was funded under NIH grant S10OD021832. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169. SAXS data collection was also conducted at the Advanced Light Source, at the SYBLS beamline supported by NIH project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10 OD018483. Results shown in this report are derived from work performed at Structural Biology Center funded by the U.S. Department of Energy, Office of Biological and Environmental Research, and operated for the DOE Office of Science at the Advanced Photon Source by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.De novo protein design methods can create proteins with folds not yet seen in nature. These methods largely focus on optimizing the compatibility between the designed sequence and the intended conformation, without explicit consideration of protein folding pathways. Deeply knotted proteins, whose topologies may introduce substantial barriers to folding, thus represent an interesting test case for protein design. Here we report our attempts to design proteins with trefoil (31) and pentafoil (51) knotted topologies. We extended previously described algorithms for tandem repeat protein design in order to construct deeply knotted backbones and matching designed repeat sequences (N = 3 repeats for the trefoil and N = 5 for the pentafoil). We confirmed the intended conformation for the trefoil design by X ray crystallography, and we report here on this protein's structure, stability, and folding behaviour. The pentafoil design misfolded into an asymmetric structure (despite a 5-fold symmetric sequence); two of the four repeat-repeat units matched the designed backbone while the other two diverged to form local contacts, leading to a trefoil rather than pentafoil knotted topology. Our results also provide insights into the folding of knotted proteins

De novo design of knotted tandem repeat proteins

Acknowledgements: We thank Meredith Steward for assistance with biochemical experiments. Support for this work was provided by the Fred Hutchinson Cancer Center and by the National Institute for General Medical Sciences (NIGMS) for both BLS (R01 GM139752) and PB (R35 GM141457). L.F.M. was supported by a Human Frontier Science Program Cross Disciplinary Fellowship (LT000395/2020-C) and an EMBO Non-Stipendiary Fellowship (ALTF 1047-2019). Crystallographic data collection was conducted at the Advanced Light Source (ALS) at the Berkeley Center for Structural Biology which is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231. The Pilatus detector at Beamline 5.0.1 (where the data was collected) was funded under NIH grant S10OD021832. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169. SAXS data collection was also conducted at the Advanced Light Source, at the SYBLS beamline supported by NIH project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10 OD018483. Results shown in this report are derived from work performed at Structural Biology Center funded by the U.S. Department of Energy, Office of Biological and Environmental Research, and operated for the DOE Office of Science at the Advanced Photon Source by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.De novo protein design methods can create proteins with folds not yet seen in nature. These methods largely focus on optimizing the compatibility between the designed sequence and the intended conformation, without explicit consideration of protein folding pathways. Deeply knotted proteins, whose topologies may introduce substantial barriers to folding, thus represent an interesting test case for protein design. Here we report our attempts to design proteins with trefoil (31) and pentafoil (51) knotted topologies. We extended previously described algorithms for tandem repeat protein design in order to construct deeply knotted backbones and matching designed repeat sequences (N = 3 repeats for the trefoil and N = 5 for the pentafoil). We confirmed the intended conformation for the trefoil design by X ray crystallography, and we report here on this protein’s structure, stability, and folding behaviour. The pentafoil design misfolded into an asymmetric structure (despite a 5-fold symmetric sequence); two of the four repeat-repeat units matched the designed backbone while the other two diverged to form local contacts, leading to a trefoil rather than pentafoil knotted topology. Our results also provide insights into the folding of knotted proteins

Genetically Encoded XTEN‐based Hydrogels with Tunable Viscoelasticity and Biodegradability for Injectable Cell Therapies

Abstract While direct cell transplantation holds great promise in treating many debilitating diseases, poor cell survival and engraftment following injection have limited effective clinical translation. Though injectable biomaterials offer protection against membrane‐damaging extensional flow and supply a supportive 3D environment in vivo that ultimately improves cell retention and therapeutic costs, most are created from synthetic or naturally harvested polymers that are immunogenic and/or chemically ill‐defined. This work presents a shear‐thinning and self‐healing telechelic recombinant protein‐based hydrogel designed around XTEN – a well‐expressible, non‐immunogenic, and intrinsically disordered polypeptide previously evolved as a genetically encoded alternative to PEGylation to “eXTENd” the in vivo half‐life of fused protein therapeutics. By flanking XTEN with self‐associating coil domains derived from cartilage oligomeric matrix protein, single‐component physically crosslinked hydrogels exhibiting rapid shear thinning and self‐healing through homopentameric coiled‐coil bundling are formed. Individual and combined point mutations that variably stabilize coil association enables a straightforward method to genetically program material viscoelasticity and biodegradability. Finally, these materials protect and sustain viability of encapsulated human fibroblasts, hepatocytes, embryonic kidney (HEK), and embryonic stem‐cell‐derived cardiomyocytes (hESC‐CMs) through culture, injection, and transcutaneous implantation in mice. These injectable XTEN‐based hydrogels show promise for both in vitro cell culture and in vivo cell transplantation applications

Precisely patterned nanofibres made from extendable protein multiplexes

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha-helical coiled coils have such symmetric, extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibres can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibres can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches

De novo design of protein logic gates

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions

Blueprinting extendable nanomaterials with standardized protein blocks

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints

Motivation for Participation in Campus Recreation Based on Activity Type

In this study, we examined differences in leisure motivation on the basis of the type of campus recreational activity in which a student was engaged. Specifically, campus recreational activities included aquatics, group fitness, intramural sports, informal sports, and informal fitness. The Leisure Motivation Scale (Beard and Ragheb, 1983) was completed by subjects after they engaged in a campus recreation activity. A 95% confidence level was established a priori, but a Bonferroni adjustment resulted in α ≤ .0015. Results from an analysis of variance indicated differences between activity type. Subjects engaged in informal sports were less motivated by competency/mastery factors than those engaged in other campus recreation activities. In addition, stimulus avoidance was a less predominant motivator in subjects participating in intramural sports than in those involved in other types of programs. These results suggest that students engaged in different activities are motivated by different factors and have implications for campus recreation programmers and marketers as they design programs