Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microscope enables a direct comparison between features observed in the fluorescence microscope and those observed in the images obtained with the AFM, in air or under liquid. The cracks in polymerized Langmuir-Blodgett films of lO,l2-pentacosadiynoic acid as observed in the fluorescence microscope run parallel to one of the lattice directions of the crystal as revealed by molecular resolution images obtained with the AFM. The orientation of these cracks also coincides with the polarization direction of the fluorescent light, indicating that the cracks run along the polymer backbone. Ripple-like corrugations on a submicrometer scale have been observed, which may be due to mechanical stress created during the polymerization process

Protein-DNA force assay in a microfluidic format

The detailed study of protein-DNA interactions is a core effort to elucidate physiological processes, including gene regulation, DNA repair and the immune response. The molecular force assay (MFA) is an established method to study DNA-binding proteins. In particular, high-affinity binder dissociation is made possible by the application of force. Microfluidic lab-on-a-chip approaches have proven helpful for parallelization, small sample volumes, reproducibility, and low cost. We report the successful combination of these two principles, forming a microfluidic molecular force assay and representing a novel use for the established MITOMI chip design. We present, characterize, validate and apply this integrated method. An alternative confocal fluorescence microscopy readout and analysis method is introduced and validated. In a multiplexing application, EcoRI binding is detected and characterized. This method paves the way for quantitative on-chip force measurements. It is suited for integration with DNA micro-spotting and in vitro expression of transcription factors to form a high-throughput chip for detailed DNA-protein interaction studies

Ligand Binding Stabilizes Cellulosomal Cohesins as Revealed by AFM-based Single-Molecule Force Spectroscopy

The cohesin-dockerin receptor-ligand family is the key element in the formation of multi-enzyme lignocellulose-digesting extracellular complexes called cellulosomes. Changes in a receptor protein upon binding of a ligand - commonly referred to as allostery - are not just essential for signalling, but may also alter the overall mechanical stability of a protein receptor. Here, we measured the change in mechanical stability of a library of cohesin receptor domains upon binding of their dockerin ligands in a multiplexed atomic force microscopy-based single-molecule force spectroscopy experiment. A parallelized, cell-free protein expression and immobilization protocol enables rapid mechanical phenotyping of an entire library of constructs with a single cantilever and thus ensures high throughput and precision. Our results show that dockerin binding increases the mechanical stability of every probed cohesin independently of its original folding strength. Furthermore, our results indicate that certain cohesins undergo a transition from a multitude of different folds or unfolding pathways to a single stable fold upon binding their ligand

Biasing effects of receptor-ligand complexes on protein-unfolding statistics

Protein receptor-ligand pairs are increasingly used as specific molecular handles in single-molecule protein-unfolding experiments. Further, known marker domains, also referred to as fingerprints, provide unique unfolding signatures to identify specific single-molecule interactions, when receptor-ligand pairs themselves are investigated. We show here that in cases where there is an overlap between the probability distribution associated with fingerprint domain unfolding and that associated with receptor-ligand dissociation, the experimentally measured force distributions are mutually biased. This biasing effect masks the true parameters of the underlying free energy landscape. To address this, we present a model-free theoretical framework that corrects for the biasing effect caused by such overlapping distributions

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

Single‐Molecule Manipulation in Zero‐Mode Waveguides

The mechanobiology of receptor–ligand interactions and force‐induced enzymatic turnover can be revealed by simultaneous measurements of force response and fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic force microscopy (AFM). A fully automatized workflow is established to manipulate single molecules inside ZMWs autonomously with noninvasive cantilever tip localization. A protein model system comprising a receptor–ligand pair of streptavidin blocked with a biotin‐tagged ligand is introduced. The ligand is pulled out of streptavidin by an AFM cantilever leaving the receptor vacant for reoccupation by freely diffusing fluorescently labeled biotin, which can be detected in single‐molecule fluorescence concurrently to study rebinding rates. This work illustrates the potential of the seamless fusion of these two powerful single‐molecule techniques

Parallel Force Assay for Protein-Protein Interactions

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay

Parallel Force Assay for Protein-Protein Interactions

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay

Atomic force microscopy-based mechanobiology

Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer ReviewedPostprint (published version

A Force-Based, Parallel Assay for the Quantification of Protein-DNA Interactions

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA),parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction