Connecting biochemistry and elelctronics with artifical allosteric protein biosensors

The idea that biological systems can be built from standard components is the central tenet of Synthetic Biology. Although proteins control most of real time information and energy flow in a cell, our ability to create protein-based switches and thereon based circuits is woefully underdeveloped. We addressed this by developing a generally applicable strategy for converting constitutively active enzymes into allosterically controlled switches. This was achieved by constructing chimeric enzymes where the enzymatically active part is fused with a conformational switch. We demonstrated that such basic signaling units could be compiled into higher order biosensor systems capable of detecting potentially any analyte. Specifically using this approach we constructed electrochemical and optical biosensors specific to ions, small molecule drugs and proteins

Copper-free sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry

Generation of efficient fingerprint for GFP-like fold and computational identification of potential GFP-like homologs

There is a considerable interest in the detection of GFP-like proteins due to their structural stability and functional usefulness. GFP-like proteins share highly conserved beta-barrel fold with 11 beta-strands. However, their low sequence identity hampers efficient identification of their homologous proteins from database. In this study, an attempt was made to generate a fingerprint for efficient detection of GFP-like proteins. Overlapped conserved residues (OCR)-based approach has been used to design a protein fingerprint based on sequentially and structurally conserved residues in secondary structures to detect homologous proteins very efficiently. Therefore, a fingerprint for GFP-like fold was designed using the OCR approach. However, its specificity was too low to be used for the identification of novel proteins. The conserved residues of loop regions were added and optimized to improve its specificity without losing its high sensitivity. The optimized fingerprint was employed to scan NR database. A total of 20 hypothetical proteins were detected, among which nine were validated as potential GFP-like homologs

Control of allosteric electrochemical protein switch using magnetic signals

We report a novel approach for magneto-controlled activation of an artificial electro-enzymatic cascade. The input signal triggers release of a caged ligand peptide, its proteolytic processing and activation of an artificial allosteric enzyme based on PQQ-dependent glucose dehydrogenase. The developed cascade was used to assemble a magneto-controlled biofuel cell

Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues

Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S,4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 °C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions

Control of Allosteric Protein Electrochemical Switches with Biomolecular and Electronic Signals

The construction of allosteric protein switches is a key goal of synthetic biology. Such switches can be compiled into signaling systems mimicking information and energy processing systems of living organisms. Here we demonstrate construction of a biocatalytic electrode functionalized with a recombinant chimeric protein between pyrroloquinoline quinone-dependent glucose dehydrogenase and calmodulin. This electrode could be activated by calmodulin-binding peptide and showed a high bioelectrocatalytic current (ca. 300 μA) due to efficient direct electron transfer. In order to expand the types of inputs that can be used to activate the developed electrode, we constructed a caged version of calmodulin-binding peptide that could be proteolytically uncaged using a protease of choice. Finally, the complexity of the switchable bioelectrochemical system was further increased by the use of almost any kind of molecule/biomolecule or electronic signal, unequivocally proving the orthogonality of the aforementioned system

Redesigning of anti-c-Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition

In vivo production of functional single-chain Fv fragment with an N-terminal-specific bio-orthogonal reactive group

Just where you want it: Grafting target scFv complementarity-determining regions onto stable frameworks (FR) and consensus-based engineering of methionines on the FR enables the production of methionine-free scFv that can be specifically modified with a bio-orthogonal reactive group on its N-terminal through in vivo incorporation of a methionine analogue

Computational screening of potential non-immunoglobulin scaffolds using overlapped conserved residues (OCR)-based fingerprints

Cystatins and lipocalins have attracted considerable interest for their potential applications in non-immunoglobulin protein scaffold engineering. In the present study, their potential homologs were screened computationally from non-redundant protein sequence database based on the overlapped conserved residues (OCR)-fingerprints, which can detect the protein family with low sequence identity, such as cystatins and lipocalins. Two types of OCR-fingerprints for each family were designed and showed very high detection efficiency (>90%). The protein sequence database was scanned by the fingerprints, which yielded the hypothetical sequences for cystatins and lipocalins. The hypothetical sequences were validated further based on their sequence motifs and structural models, which allowed an identification of the potential homologs of cystatins and lipocalins