Living materials with programmable functionalities grown from engineered microbial co-cultures.

Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically engineered organisms. Here, we describe an approach to fabricating functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulose-based engineered living materials with potential applications in biosensing and biocatalysis.

Data-driven enzyme immobilisation: a case study using DNA to immobilise galactose oxidase.

Biocatalysis has the potential to enable green chemistry. New methods of enzyme immobilisation will be required to improve enzyme stability, product purification, and compatibility of different enzymes in the same reaction conditions. Deoxyribonucleic acid (DNA) stands out among supramolecular scaffolds, as simple Watson-Crick base-pairing rules can be used to rationally design a unique nanoscale environment around each individual enzyme in a cascade. Enhancements of enzyme activity and stability on DNA nanostructures have previously been reported, but never in the context of industrially relevant chemical syntheses or reaction conditions. Here, the authors show DNA can enhance the activity and stability of a galactose oxidase mutant, which could be used in a cascade to produce bioplastics from lignin. The enzyme was enhanced in the cell-free extract, which to their knowledge has not been shown before for any enzymes on DNA. This is significant because crude biocatalytic reactions are vastly more cost-effective. This opens the door to further work on multienzyme cascades by tuning the properties of individual enzymes.

The free energy landscape of retroviral integration.

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

Enzyme-Mediated, Site-Specific Protein Coupling Strategies for Surface-Based Binding Assays.

Covalent surface immobilization of proteins for binding assays is typically performed non-specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4'-phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site-specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors' functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor-ligand pairs.

Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics.

Cellulosomes are polyprotein machineries that efficiently degrade cellulosic material. Crucial to their function are scaffolds consisting of highly homologous cohesin domains, which serve a dual role by coordinating a multiplicity of enzymes as well as anchoring the microbe to its substrate. Here we combined two approaches to elucidate the mechanical properties of the main scaffold ScaA of Acetivibrio cellulolyticus. A newly developed parallelized one-pot in vitro transcription-translation and protein pull-down protocol enabled high-throughput atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) measurements of all cohesins from ScaA with a single cantilever, thus promising improved relative force comparability. Albeit very similar in sequence, the hanging cohesins showed considerably lower unfolding forces than the bridging cohesins, which are subjected to force when the microbe is anchored to its substrate. Additionally, all-atom steered molecular dynamics (SMD) simulations on homology models offered insight into the process of cohesin unfolding under force. Based on the differences among the individual force propagation pathways and their associated correlation communities, we designed mutants to tune the mechanical stability of the weakest hanging cohesin. The proposed mutants were tested in a second high-throughput AFM SMFS experiment revealing that in one case a single alanine to glycine point mutation suffices to more than double the mechanical stability. In summary, we have successfully characterized the force induced unfolding behavior of all cohesins from the scaffoldin ScaA, as well as revealed how small changes in sequence can have large effects on force resilience in cohesin domains. Our strategy provides an efficient way to test and improve the mechanical integrity of protein domains in general.

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox.

Single-molecule force spectroscopy sheds light onto the free energy landscapes governing protein folding and molecular recognition. Since only a single molecule or single molecular complex is probed at any given point in time, the technique is capable of identifying low-probability conformations within a large ensemble of possibilities. It furthermore allows choosing certain unbinding pathways through careful selection of the points at which the force acts on the protein or molecular complex. This review focuses on recent innovations in construct design, site-specific bioconjugation, measurement techniques, instrumental advances, and data analysis methods for improving workflow, throughput, and data yield of AFM-based single-molecule force spectroscopy experiments. Current trends that we highlight include customized fingerprint domains, peptide tags for site-specific covalent surface attachment, and polyproteins that are formed through mechanostable receptor-ligand interactions. Recent methods to improve measurement stability, signal-to-noise ratio, and force precision are presented, and theoretical considerations, analysis methods, and algorithms for analyzing large numbers of force-extension curves are further discussed. The various innovations identified here will serve as a starting point to researchers in the field looking for opportunities to push the limits of the technique further.

Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles.

Single-molecule force spectroscopy greatly benefits from site-specific surface immobilization and specific probing with a functionalized cantilever. Here, we describe a streamlined approach to such experiments by covalently attaching mechanically stable receptors onto proteins of interest (POI) to improve pickup efficiency and specificity. This platform provides improved throughput, allows precise control over the pulling geometry, and allows for multiple constructs to be probed with the same ligand-modified cantilever. We employ two orthogonal enzymatic ligation reactions [sortase and phosphopantetheinyl transferase (Sfp)] to covalently immobilize POI to a pegylated surface and to subsequently ligate the POI to a mechanically stable dockerin domain at the protein's C-terminus for use as a high-strength pulling handle. Our configuration permits expression and folding of the POI to proceed independently from the mechanically stable receptor used for specific probing and requires only two short terminal peptide sequences (i.e., ybbR-tag and sortase C-tag). We applied this system successfully to proteins expressed using in vitro transcription and translation reactions without a protein purification step and to purified proteins expressed in Escherichia coli.

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Mapping Mechanical Force Propagation through Biomolecular Complexes.

Here we employ single-molecule force spectroscopy with an atomic force microscope (AFM) and steered molecular dynamics (SMD) simulations to reveal force propagation pathways through a mechanically ultrastable multidomain cellulosome protein complex. We demonstrate a new combination of network-based correlation analysis supported by AFM directional pulling experiments, which allowed us to visualize stiff paths through the protein complex along which force is transmitted. The results implicate specific force-propagation routes nonparallel to the pulling axis that are advantageous for achieving high dissociation forces.

Ultrastable cellulosome-adhesion complex tightens under load.

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass.

From genes to protein mechanics on a chip.

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

Improving the NADH-cofactor specificity of the highly active AdhZ3 and AdhZ2 from Escherichia coli K-12.

Biocatalysis is a promising tool for the sustainable production of chemicals. When cofactor depending enzymatic reactions are involved the applicability of the right cofactor is a central issue. One important example in this regard is the production of alcohols by nicotinamide cofactor (NAD(P)(+)) depending alcohol dehydrogenases. AdhZ3 from Escherichia coli, which is important for the production of alcohols from biomass, has a preference for NADPH as cofactor. We used a structure guided site-specific random approach, to change the cofactor preference towards NADH and to deduce more general rules for redesigning the cofactor specificity. Transfer of a triplet motif from NADH preferring horse liver ADH to AdhZ3 showed an insufficient switch in the preference towards NADH. A combinatorial site saturation mutagenesis altering three residues at once was applied. Library screening with two different cofactor concentrations (0.1 and 0.3mM) resulted in nine improved variants with AdhZ3-LND having the highest vmax and AdhZ3-CND having the lowest K(m). Asparagine was the most frequent amino acid found in eight of nine triplet motifs. To verify the triplet-motif, two variants of E. coli AdhZ2 DIN and LND were designed and confirmed for improved activity with NADH.