Abstract:Biomaterials based on immobilized proteins are key elements of many biomedical and industrial technologies. However, applications are limited by an inability to precisely construct materials of high homogeneity and defined content. We present here a general "protein-limited immobilization" strategy by combining the rapid, bioorthogonal, and biocompatible properties of a tetrazine-strained trans-cyclooctene reaction with genetic code expansion to sitespecifically place the tetrazine into a protein. For the firs… Show more
“…The IEDDA reaction is a [4+2] cycloaddition that occurs between an electron-deficient diene such as a 1,2,4,5-tetrazine and an electron-rich dienophile such as a strained alkene 1 . Bioorthogonal IEDDA reactions, such as those first described by Fox and colleagues, can reach rates upwards of 10 6 M -1 s -1 , allowing complete reaction within minutes at sub-micromolar concentrations 1,9,[11][12][13][14] . Amino acid derivatives containing azide, cyclopropene, alkyne, trans-cyclooctene (TCO), and tetrazine functionalities have all been genetically encoded into proteins 15 , albeit not yet in a manner conducive to dual encoding and subsequent intracellular labeling in vivo.…”
The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.
“…The IEDDA reaction is a [4+2] cycloaddition that occurs between an electron-deficient diene such as a 1,2,4,5-tetrazine and an electron-rich dienophile such as a strained alkene 1 . Bioorthogonal IEDDA reactions, such as those first described by Fox and colleagues, can reach rates upwards of 10 6 M -1 s -1 , allowing complete reaction within minutes at sub-micromolar concentrations 1,9,[11][12][13][14] . Amino acid derivatives containing azide, cyclopropene, alkyne, trans-cyclooctene (TCO), and tetrazine functionalities have all been genetically encoded into proteins 15 , albeit not yet in a manner conducive to dual encoding and subsequent intracellular labeling in vivo.…”
The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.
“…As shown in Figure 1A, these constructs, referred to hereafter as CA AzF , CA Cys , and CA Tet , were designed to permit site-specific ligation to either DBCO, maleimide, or sTCO-functionalized silica microparticles (1 ÎŒm mean diameter), via slow, intermediate or fast ligation reaction rate constant, respectively. Residue position 126 was chosen because it is remote from the active site, which would reduce the probability that immobilization would result in obstruction of the active site 12 . Importantly, to generate CA AzF , CA Cys , and CA Tet , we used the thermally stable variant C205S in which the only native cysteine in CA was replaced with a serine.…”
Section: Impact Of Ligation Efficiency On Ensemble Activity and Structurementioning
confidence: 99%
“…Cloning, Expression, and Purification of CA constructs CA constructs (Table S1) were cloned from a C205S thermal stable variant using methods that were previously described elsewhere 12 . Following cloning, CA constructs were produced using the DEAL approach as we've previously described 35 .…”
Section: Surface Preparation and Characterizationmentioning
confidence: 99%
“…Additionally, the promise of such biohybrid materials is frequently limited by the fact that enzymes generally function in only a narrow range of conditions. As such, the development of strategies to improve the functional utility of enzymes upon immobilization has been the focus of considerable effort [9][10][11][12] .…”
During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus probability of immobilization with reactive surface sites (i.e., ligation efficiency). Here, the connection between ligation reaction efficiency and the retention of enzyme structure and activity was investigated by leveraging the extremely fast reaction of strained trans-cycopropenecyclooctenes (sTCOs) and tetrazines (Tet). Remarkably, upon immobilization via Tet-sTCO chemistry, carbonic anhydrase (CA) retained 77% of its solution-phase activity, while immobilization via less efficient reaction chemistries, such as thiol-maleimide and azide-dibenzocyclooctyne, led to activity retention of only 46% and 27%, respectively. Dynamic single-molecule (SM) fluorescence tracking methods further revealed that longer surface search distances prior to immobilization (>0.5 ÎŒm) dramatically increased the probability of CA unfolding. Notably, CA distance to immobilization was significantly reduced through the use of tetrazine-sTCO chemistry, which correlated with the increased retention of structure and activity of immobilized CA compared to the use of slower ligation chemistries. These findings provide unprecedented insight into the role of ligation reaction efficiency in mediating the exploration denaturing hotspots on surfaces by enzymes, which, in turn, may have major ramifications in the creation of functional biohybrid materials.
“…Understanding the behavior of surface-adsorbed proteins on nanoparticles (NP) is crucial when NPs are used in vivo as a therapeutic drug carrier, phototherapeutic agent, or imaging tool [1][2][3][4] . There is increasing evidence that the composition of the adsorbed proteins is not as important as their structure and orientation [5][6][7] , as the latter governs the three-dimensional presentation of proteins for molecular recognition, which will define the biological performance and fate of the NP [8][9][10] . For example, the arrangement and orientation of the adsorbed proteins on a NP define the distribution of epitopes presented for immune response 5 and determine the success of cell targeting 6,9 .…”
The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticle's bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo.
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