Protein Interaction Kinetics Delimit the Performance of Phosphorylation-Driven Protein Switches.

Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo-designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between "on" and "off" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.

Information Transmission through Biotic-Abiotic Interfaces to Restore or Enhance Human Function.

Advancements in reliable information transfer across biotic-abiotic interfaces have enabled the restoration of lost human function. For example, communication between neuronal cells and electrical devices restores the ability to walk to a tetraplegic patient and vision to patients blinded by retinal disease. These impactful medical achievements are aided by tailored biotic-abiotic interfaces that maximize information transfer fidelity by considering the physical properties of the underlying biological and synthetic components. This Review develops a modular framework to define and describe the engineering of biotic and abiotic components as well as the design of interfaces to facilitate biotic-abiotic information transfer using light or electricity. Delineating the properties of the biotic, interface, and abiotic components that enable communication can serve as a guide for future research in this highly interdisciplinary field. Application of synthetic biology to engineer light-sensitive proteins has facilitated the control of neural signaling and the restoration of rudimentary vision after retinal blindness. Electrophysiological methodologies that use brain-computer interfaces and stimulating implants to bypass spinal column injuries have led to the rehabilitation of limb movement and walking ability. Cellular interfacing methodologies and on-chip learning capability have been made possible by organic transistors that mimic the information processing capacity of neurons. The collaboration of molecular biologists, material scientists, and electrical engineers in the emerging field of biotic-abiotic interfacing will lead to the development of prosthetics capable of responding to thought and experiencing touch sensation via direct integration into the human nervous system. Further interdisciplinary research will improve electrical and optical interfacing technologies for the restoration of vision, offering greater visual acuity and potentially color vision in the near future.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

Fabrication of Electronically Conductive Protein-Heme Nanowires for Power Harvesting.

Electronically conductive protein-based materials can enable the creation of bioelectronic components and devices from sustainable and nontoxic materials, while also being well-suited to interface with biological systems, such as living cells, for biosensor applications. However, as proteins are generally electrical insulators, the ability to render protein assemblies electroactive in a tailorable manner can usher in a plethora of useful materials. Here, an approach to fabricate electronically conductive protein nanowires is presented by aligning heme molecules in proximity along protein filaments, with these nanowires also possessing charge transfer abilities that enable energy harvesting from ambient humidity. The heme-incorporated protein nanowires demonstrate electron transfer over micrometer distances, with conductive atomic force microscopy showing individual nanowires having comparable conductance to other previously characterized heme-based bacterial nanowires. Exposure of multilayer nanowire films to humidity produces an electrical current, presumably through water molecules ionizing carboxyl groups in the filament and creating an unbalanced total charge distribution that is enhanced by the heme. Incorporation of heme and potentially other metal-center porphyrin molecules into protein nanostructures could pave the way for structurally- and electrically-defined protein-based bioelectronic devices.

Effects of Phosphorylation on the Activity, Inhibition and Stability of Carbonic Anhydrases.

Carbonic anhydrases (CAs) are a metalloenzyme family that have important roles in cellular processes including pH homeostasis and have been implicated in multiple pathological conditions. Small molecule inhibitors have been developed to target carbonic anhydrases, but the effects of post-translational modifications (PTMs) on the activity and inhibition profiles of these enzymes remain unclear. Here, we investigate the effects of phosphorylation, the most prevalent carbonic anhydrase PTM, on the activities and drug-binding affinities of human CAI and CAII, two heavily modified active isozymes. Using serine to glutamic acid (S > E) mutations to mimic the effect of phosphorylation, we demonstrate that phosphomimics at a single site can significantly increase or decrease the catalytic efficiencies of CAs, depending on both the position of the modification and the CA isoform. We also show that the S > E mutation at Ser50 of hCAII decreases the binding affinities of hCAII with well-characterized sulphonamide inhibitors including by over 800-fold for acetazolamide. Our findings suggest that CA phosphorylation may serve as a regulatory mechanism for enzymatic activity, and affect the binding affinity and specificity of small, drug and drug-like molecules. This work should motivate future studies examining the PTM-modification forms of CAs and their distributions, which should provide insights into CA physiopathological functions and facilitate the development of 'modform-specific' carbonic anhydrase inhibitors.

A versatile multimodal chromatography strategy to rapidly purify protein nanostructures assembled in cell lysates.

BACKGROUND: Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. RESULTS: We developed an efficient approach to purify functionalized protein nanostructures directly from bacterial lysates through a type of multimodal chromatography (MMC) that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. We employed the ultrastable filamentous protein gamma-prefoldin as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using MMC was tested across a wide range of salt concentrations and pH, demonstrating that the MMC resin is robust, however the optimal choice of salt species, salt concentration, and pH is likely dependent on the protein nanostructure to be purified. In addition, we show that pre-processing of the samples with tangential flow filtration to remove nucleotides and metabolites improves resin capacity, and that post-processing with Triton X-114 phase partitioning is useful to remove lipids and any remaining lipid-associated protein. Subsequently, functionalized protein filaments were purified from bacterial lysates using MMC and shown to be free of unincorporated subunits. The assembly and purification of protein filaments with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Förster resonance energy transfer, and transmission electron microscopy. Finally, we compared our MMC workflow to anion exchange chromatography with the purification of encapsulin nanocompartments containing a fluorescent protein as a cargo, demonstrating the versatility of the protocol and that the purity of the assembly is comparable to more traditional procedures. CONCLUSIONS: We envision that the use of MMC will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.

Harnessing the Structural and Functional Diversity of Protein Filaments as Biomaterial Scaffolds.

The natural ability of many proteins to polymerize into highly structured filaments has been harnessed as scaffolds to align functional molecules in a diverse range of biomaterials. Protein-engineering methodologies also enable the structural and physical properties of filaments to be tailored for specific biomaterial applications through genetic engineering or filaments built from the ground up using advances in the computational prediction of protein folding and assembly. Using these approaches, protein filament-based biomaterials have been engineered to accelerate enzymatic catalysis, provide routes for the biomineralization of inorganic materials, facilitate energy production and transfer, and provide support for mammalian cells for tissue engineering. In this review, we describe how the unique structural and functional diversity in natural and computationally designed protein filaments can be harnessed in biomaterials. In addition, we detail applications of these protein assemblies as material scaffolds with a particular emphasis on applications that exploit unique properties of specific filaments. Through the diversity of protein filaments, the biomaterial engineer's toolbox contains many modular protein filaments that will likely be incorporated as the main structural component of future biomaterials.

Multiplexed Screening of Thousands of Natural Products for Protein-Ligand Binding in Native Mass Spectrometry.

The structural diversity of natural products offers unique opportunities for drug discovery, but challenges associated with their isolation and screening can hinder the identification of drug-like molecules from complex natural product extracts. Here we introduce a mass spectrometry-based approach that integrates untargeted metabolomics with multistage, high-resolution native mass spectrometry to rapidly identify natural products that bind to therapeutically relevant protein targets. By directly screening crude natural product extracts containing thousands of drug-like small molecules using a single, rapid measurement, we could identify novel natural product ligands of human drug targets without fractionation. This method should significantly increase the efficiency of target-based natural product drug discovery workflows.

Functional Applications of Nucleic Acid-Protein Hybrid Nanostructures.

Combining the diverse chemical functionality of proteins with the predictable structural assembly of nucleic acids has enabled the creation of hybrid nanostructures for a range of biotechnology applications. Through the attachment of proteins onto or within nucleic acid nanostructures, materials with dynamic capabilities can be created that include switchable enzyme activity, targeted drug delivery, and multienzyme cascades for biocatalysis. Investigations of difficult-to-study biological mechanisms have also been aided by using DNA-protein assemblies that mimic natural processes in a controllable manner. Furthermore, advances that enable the recombinant production and intracellular assembly of hybrid nanostructures have the potential to overcome the significant manufacturing cost that has limited the use of DNA and RNA nanotechnology.

Design of Tunable Protein Interfaces Controlled by Post-Translational Modifications.

The design of protein interaction interfaces is a cornerstone of synthetic biology, where they can be used to promote the association of protein subunits into active molecular complexes or into protein nanostructures. In nature, protein interactions can be modulated by post-translational modifications (PTMs) that modify the protein interfaces with the addition and removal of various chemical groups. PTMs thus represent a means to gain control over protein interactions, yet they have seldom been considered in the design of synthetic proteins. Here, we explore the potential of a reversible PTM, serine phosphorylation, to modulate the interactions between peptides. We designed a series of interacting peptide pairs, including heterodimeric coiled coils, that contained one or more protein kinase A (PKA) recognition motifs. Our set of peptide pairs comprised interactions ranging from nanomolar to micromolar affinities. Mass spectrometry analyses showed that all peptides were excellent phosphorylation substrates of PKA, and subsequent phosphate removal could be catalyzed by lambda protein phosphatase. Binding kinetics measurements performed before and after treatment of the peptides with PKA revealed that phosphorylation of the target serines affected both the association and dissociation rates of the interacting peptides. We observed both the strengthening of interactions (up to an 11-fold decrease in Kd) and the weakening of interactions (up to a 180-fold increase in Kd). De novo-designed PTM-modulated interfaces will be useful to control the association of proteins in biological systems using protein-modifying enzymes, expanding the paradigm of self-assembly to encompass controlled assembly of engineerable protein complexes.

Structural Determination of a Filamentous Chaperone to Fabricate Electronically Conductive Metalloprotein Nanowires.

The transfer of electrons through protein complexes is central to cellular respiration. Exploiting proteins for charge transfer in a controllable fashion has the potential to revolutionize the integration of biological systems and electronic devices. Here we characterize the structure of an ultrastable protein filament and engineer the filament subunits to create electronically conductive nanowires under aqueous conditions. Cryoelectron microscopy was used to resolve the helical structure of gamma-prefoldin, a filamentous protein from a hyperthermophilic archaeon. Conjugation of tetra-heme c3-type cytochromes along the longitudinal axis of the filament created nanowires capable of long-range electron transfer. Electrochemical transport measurements indicated networks of the nanowires capable of conducting current between electrodes at the redox potential of the cytochromes. Functionalization of these highly engineerable nanowires with other molecules, such as redox enzymes, may be useful for bioelectronic applications.

Production of Multicomponent Protein Templates for the Positioning and Stabilization of Enzymes.

Harnessing the ability of proteins to self-assemble into complex structures has enabled the creation of templates for applications in nanotechnology. Protein templates can be used to position functional molecules in regular patterns with nanometer precision over large surface areas. A difficult but successful approach to building customizable protein templates involves designing novel protein-protein interfaces to join protein building blocks into ordered arrangements. This approach was illustrated recently by engineering the protein interfaces of a molecular chaperone to produce filamentous templates composed of repeating subunits. In this chapter, we describe how these multicomponent protein templates can be produced recombinantly, assembled into filaments, and used as material templates. The templates enable the positioning and alignment of functional molecules at varying distances along the length of the filament, which can be demonstrated using a Förster resonance energy transfer (FRET) assay. In addition, we describe a method to quantify the chaperone ability of these filaments to stabilize and protect other proteins from thermal-induced aggregation-a useful property for bionanotechnology applications that involve molecular scaffolds for positioning and stabilizing enzymes.

Enhanced Enzyme Activity through Scaffolding on Customizable Self-Assembling Protein Filaments.

Precisely organized enzyme complexes are often found in nature to support complex metabolic reactions in a highly efficient and specific manner. Scaffolding enzymes on artificial materials has thus gained attention as a promising biomimetic strategy to design biocatalytic systems with enhanced productivity. Herein, a versatile scaffolding platform that can immobilize enzymes on customizable nanofibers is reported. An ultrastable self-assembling filamentous protein, the gamma-prefoldin (γ-PFD), is genetically engineered to display an array of peptide tags, which can specifically and stably bind enzymes containing the counterpart domain through simple in vitro mixing. Successful immobilization of proteins along the filamentous template in tunable density is first verified using fluorescent proteins. Then, two different model enzymes, glucose oxidase and horseradish peroxidase, are used to demonstrate that scaffold attachment could enhance the intrinsic catalytic activity of the immobilized enzymes. Considering the previously reported ability of γ-PFD to bind and stabilize a broad range of proteins, the filament's interaction with the bound enzymes may have created a favorable microenvironment for catalysis. It is envisioned that the strategy described here may provide a generally applicable methodology for the scaffolded assembly of multienzymatic complexes for use in biocatalysis.

Engineering bioorthogonal protein-polymer hybrid hydrogel as a functional protein immobilization platform.

We demonstrate the synthesis of protein-polymer hybrid hydrogel that can be used as a platform for immobilizing functional proteins. Orthogonal chemistry was employed for cross-linking the hybrid network and conjugating proteins to the gel backbone, allowing for the convenient, one-pot formation of a functionalized hydrogel. The resulting hydrogel had tunable mechanical properties, was stable in solution, and biocompatible.

Prefoldins in Archaea.

Molecular chaperones promote the correct folding of proteins in aggregation-prone cellular environments by stabilizing nascent polypeptide chains and providing appropriate folding conditions. Prefoldins (PFDs) are molecular chaperones found in archaea and eukaryotes, generally characterized by a unique jellyfish-like hexameric structure consisting of a rigid beta-barrel backbone with protruding flexible coiled-coils. Unlike eukaryotic PFDs that mainly interact with cytoskeletal components, archaeal PFDs can stabilize a wide range of substrates; such versatility reflects PFD's role as a key element in archaeal chaperone systems, which often lack general nascent-chain binding chaperone components such as Hsp70. While archaeal PFDs mainly exist as hexameric complexes, their structural diversity ranges from tetramers to filamentous oligomers. PFDs bind and stabilize nonnative proteins using varying numbers of coiled-coils, and subsequently transfer the substrate to a group II chaperonin (CPN) for refolding. The distinct structure and specific function of archaeal PFDs have been exploited for a broad range of applications in biotechnology; furthermore, a filament-forming variant of PFD has been used to fabricate nanoscale architectures of defined shapes, demonstrating archaeal PFDs' potential applicability in nanotechnology.

Assembly of Multicomponent Protein Filaments Using Engineered Subunit Interfaces.

Exploiting the ability of proteins to self-assemble into architectural templates may provide novel routes for the positioning of functional molecules in nanotechnology. Here we report the engineering of multicomponent protein templates composed of distinct monomers that assemble in repeating orders into a dynamic functional structure. This was achieved by redesigning the protein-protein interfaces of a molecular chaperone with helical sequences to create unique subunits that assemble through orthogonal coiled-coils into filaments up to several hundred nanometers in length. Subsequently, it was demonstrated that functional proteins could be fused to the subunits to achieve ordered alignment along filaments. Importantly, the multicomponent filaments had molecular chaperone activity and could prevent other proteins from thermal-induced aggregation, a potentially useful property for the scaffolding of enzymes. The design in this work is presented as proof-of-concept for the creation of modular templates that could potentially be used to position functional molecules, stabilize other proteins such as enzymes, and enable controlled assembly of nanostructures with unique topologies.

Controlled Assembly of the Filamentous Chaperone Gamma-Prefoldin into Defined Nanostructures.

Self-assembling protein templates have enormous potential for the fabrication of multifunctional nanostructures that require precise positioning of individual molecules, such as enzymes and inorganic moieties, in regular patterns. A recently described approach uses ultrastable filaments composed of the gamma-prefoldin (γPFD) protein and engineered connector proteins to construct novel architectures useful for basic research and practical applications in nanobiotechnology. Here we describe the production of the γPFD and connector proteins from E. coli, and the assembly of γPFD with connector proteins into macromolecular structures with defined shapes.

Protein Calligraphy: A New Concept Begins To Take Shape.

The ability to assemble molecules into supramolecular architectures of controllable size and symmetry is a long sought after goal of nanotechnology and material engineering. Proteins are particularly attractive for molecular assembly due to their inherent molecular recognition and self-assembly capabilities. Advances in the computational prediction of protein folding and quaternary assembly have enabled the design of proteins that self-assemble into complex yet predictable shapes. These protein nanostructures are opening new possibilities in biomaterials, metabolic engineering, molecular delivery, tissue engineering, and a plethora of nanomaterials. Images of protein constructs assembled from simpler structures draw comparison to characters of calligraphy. In both cases, elaborate designs emerge from basic subunits, resulting in the translation of form into function with a high degree of artistry.

Geometrical assembly of ultrastable protein templates for nanomaterials.

The fabrication of nanoscale devices requires architectural templates on which to position functional molecules in complex arrangements. Protein scaffolds are particularly promising templates for nanomaterials due to inherent molecular recognition and self-assembly capabilities combined with genetically encoded functionalities. However, difficulties in engineering protein quaternary structure into stable and well-ordered shapes have hampered progress. Here we report the development of an ultrastable biomolecular construction kit for the assembly of filamentous proteins into geometrically defined templates of controllable size and symmetry. The strategy combines redesign of protein-protein interaction specificity with the creation of tunable connector proteins that govern the assembly and projection angles of the filaments. The functionality of these nanoarchitectures is illustrated by incorporation of nanoparticles at specific locations and orientations to create hybrid materials such as conductive nanowires. These new structural components facilitate the manufacturing of nanomaterials with diverse shapes and functional properties over a wide range of processing conditions.