Design of Protein–Peptide Interaction Modules for Assembling Supramolecular Structures <i>in Vivo</i> and <i>in Vitro</i>

Synthetic biology and protein origami both require protein building blocks that behave in a reliable, predictable fashion. In particular, we require protein interaction modules with known specificity and affinity. Here, we describe three designed TRAP (Tetratricopeptide Repeat Affinity Protein)–peptide interaction pairs that are functional <i>in vivo</i>. We show that each TRAP binds to its cognate peptide and exhibits low cross-reactivity with the peptides bound by the other TRAPs. In addition, we demonstrate that the TRAP–peptide interactions are functional in many cellular contexts. In extensions of these designs, we show that the binding affinity of a TRAP–peptide pair can be systematically varied. The TRAP–peptide pairs we present thus represent a powerful set of new building blocks that are suitable for a variety of applications

Local and long-range stability in tandemly arrayed tetratricopeptide repeats

The tetratricopeptide repeat (TPR) is a 34-aa alpha-helical motif that occurs in tandem arrays in a variety of different proteins. In natural proteins, the number of TPR motifs ranges from 3 to 16 or more. These arrays function as molecular scaffolds and frequently mediate protein-protein interactions. We have shown that correctly folded TPR domain proteins, exhibiting the typical helix-turn-helix fold, can be designed by arraying tandem repeats of an idealized TPR consensus motif. To date, three designed proteins, CTPR1, CTPR2, and CTPR3 (consensus TPR number of repeats) have been characterized. Their high-resolution crystal structures show that the designed proteins indeed adopt the typical TPR fold, which is specified by the correct positioning of key residues. Here, we present a study of the thermodynamic properties and folding kinetics of this set of designed proteins. Chemical denaturation, monitored by CD and fluorescence, was used to assess the folding and global stability of each protein. NMR-detected amide proton exchange was used to investigate the stability of each construct at a residue-specific level. The results of these studies reveal a stable core, which defines the intrinsic stability of an individual TPR motif. The results also show the relationship between the number of tandem repeats and the overall stability and folding of the protei

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Probing the Molecular Origin of Native-State Flexibility in Repeat Proteins

In contrast to globular proteins, the structure of repeat proteins is dominated by a regular set of short-range interactions. This property may confer on the native state of such proteins significant elasticity. We probe the molecular origin of the spring-like behavior of repeat proteins using a designed tetratricopeptide repeat protein with three repeat units (CTPR3). Single-molecule fluorescence studies of variants of the protein with FRET pairs at different positions show a continuous expansion of the folded state of CTPR3 at low concentrations of a chemical denaturant, preceding the all-or-none transition to the unfolded state. This remarkable native-state expansion can be explained quantitatively by a reduction in the spring constant of the structure. Circular dichroism and tryptophan fluorescence spectroscopy further show that the expansion does not involve either unwinding of CTPR3 helices or unraveling of interactions within repeats. These findings point to hydrophobic inter-repeat contacts as the source of the elasticity of repeat proteins

Facile Protein Immobilization Using Engineered Surface-Active Biofilm Proteins

Immobilization of enzymes and other biomolecules to surfaces is critically important for biotechnology, with important applications in sensing and controlled delivery of molecular species for analytical or biomedical purposes. The presentation of protein recognition elements in a way that avoids denaturation and nonspecific interactions while maintaining the accessibility of the active site is a challenge for which no general solution has been found. Here we present a robust, facile method for immobilization of any protein to a surface using engineered protein building blocks. By functionalizing an interfacial protein, BslA, with peptides (SpyTag and SnoopTag) that spontaneously react with their cognate protein partners (SpyCatcher and SnoopCatcher), we are able to create patterned surfaces of protein monolayers displaying reactive tags. We demonstrate that these surfaces can be functionalized rapidly, spontaneously, and specifically with proteins of interest attached to SpyCatcher or SnoopCatcher. This method both protects the surface from nonspecific adsorption and also presents the recognition element in a uniform, active conformation. We envision that this method will have widespread applications, including immobilization of therapeutically relevant proteins for diagnostic applications

Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA

Protein adsorption and assembly at interfaces provide a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air–water and oil–water interfaces. Densely packed, high modulus monolayers form at air–water interfaces, leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil–water interfaces, leading to the production of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates from a low areal density of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small molecule surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic–lipophilic balance (HLB number) calculated based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries experiments reveal intriguing reversible and nonreversible modes of mechanical deformation, respectively. The mechanical robustness of the microcapsules and the ability to engineer their shape and to design highly specific binding responses through protein engineering suggest that these microcapsules may be useful for biomedical applications

Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA

Protein adsorption and assembly at interfaces provide a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air–water and oil–water interfaces. Densely packed, high modulus monolayers form at air–water interfaces, leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil–water interfaces, leading to the production of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates from a low areal density of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small molecule surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic–lipophilic balance (HLB number) calculated based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries experiments reveal intriguing reversible and nonreversible modes of mechanical deformation, respectively. The mechanical robustness of the microcapsules and the ability to engineer their shape and to design highly specific binding responses through protein engineering suggest that these microcapsules may be useful for biomedical applications

Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA

Protein adsorption and assembly at interfaces provide a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air–water and oil–water interfaces. Densely packed, high modulus monolayers form at air–water interfaces, leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil–water interfaces, leading to the production of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates from a low areal density of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small molecule surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic–lipophilic balance (HLB number) calculated based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries experiments reveal intriguing reversible and nonreversible modes of mechanical deformation, respectively. The mechanical robustness of the microcapsules and the ability to engineer their shape and to design highly specific binding responses through protein engineering suggest that these microcapsules may be useful for biomedical applications