Kerstin Blank

Reliable microfluidic on-chip incubation of droplets in delay-lines

Together with droplet creation, fusion and sorting, the incubation of droplets is one of the most important and essential operations for droplet-based microfluidic assays. This manuscript concerns the development of delay-lines, which are necessary to allow incubation of reactions for precise time periods. We analyze the problems associated with creating delay-lines for incubation in the minute to hour time range, which arise from back-pressure and from the dispersion in the incubation time due to the unequal speeds with which droplets pass through the delay-line. We describe delay-line systems which resolve these problems and demonstrate their use to measure reaction kinetics over several minutes in droplets.

Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments

The success of single-molecule (SM) experiments critically depends on the functional immobilization of the biomolecule(s) to be studied. With the continuing trend of combining SM fluorescence with SM force experiments, methods are required that are suitable for both types of measurements. We describe a general protocol for the site-specific and covalent coupling of any type of biomolecule that can be prepared with a free thiol group. The protocol uses a poly(ethylene glycol) (PEG) spacer, which carries an N-hydroxy succinimide (NHS) group on one end and a maleimide group on the other. After reacting the NHS group with an amino-functionalized surface, the relatively stable but highly reactive maleimide group allows the coupling of the biomolecule. This protocol provides surfaces with low fluorescence background, low nonspecific binding and a large number of reactive sites. Surfaces containing immobilized biomolecules can be obtained within 6 h.

A force-based protein biochip

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand–receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein–protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Triggering Enzymatic Activity with Force

Integrating single molecule force spectroscopy with fluorescence-based techniques allows the manipulation of an enzyme with a periodic stretching and relaxation protocol while simultaneously monitoring its catalytic activity. After releasing the stretching force we observe a higher probability for enzymatic activity at a time of 1.7 s. A detailed theoretical analysis reveals that the relaxation from the force-induced enzyme conformation to the observed active conformation follows a cascade reaction with several steps and a free energy difference of at least 8 k(B)T. Our study clearly points out the direct influence of force on enzymatic activity and opens up a new way to study and manipulate (bio)catalytic reactions at the single molecule level.

Force-based Analysis of Multidimensional Energy Landscapes: Application of Dynamic Force Spectroscopy and Steered Molecular Dynamics Simulations to an Antibody Fragment–Peptide Complex

Multidimensional energy landscapes are an intrinsic property of proteins and define their dynamic behavior as well as their response to external stimuli. In order to explore the energy landscape and its implications on the dynamic function of proteins dynamic force spectroscopy and steered molecular dynamics (SMD) simulations have proved to be important tools. In this study, these techniques have been employed to analyze the influence of the direction of the probing forces on the complex of an antibody fragment with its peptide antigen. Using an atomic force microscope, experiments were performed where the attachment points of the 12 amino acid long peptide antigen were varied. These measurements yielded clearly distinguishable basal dissociation rates and potential widths, proving that the direction of the applied force determines the unbinding pathway. Complementary atomistic SMD simulations were performed, which also show that the unbinding pathways of the system are dependent on the pulling direction. However, the main barrier to be crossed was independent of the pulling direction and is represented by a backbone hydrogen bond between Gly(H)-H40 of the antibody fragment and Glu(Oepsilon)-6(peptide) of the peptide. For each pulling direction, the observed barriers can be correlated with the rupture of specific interactions, which stabilize the bound complex. Furthermore, although the SMD simulations were performed at loading rates exceeding the experimental rates by orders of magnitude due to computational limitations, a detailed comparison of the barriers that were overcome in the SMD simulations with the data obtained from the atomic force microscope unbinding experiments show excellent agreement.

Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy

A genetically modified form of the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.

Single-biomolecule kinetics: the art of studying a single enzyme

The potential of single-enzyme studies to unravel the complex energy landscape of these polymeric catalysts is the next critical step in enzymology. From its inception in Rotmans emulsion experiments in the 1960s, the field of single-molecule enzymology has now advanced into the time-resolved age. Technological advances have enabled individual enzymatic turnover reactions to be observed with a millisecond time resolution. A number of initial studies have revealed the underlying static and dynamic disorder in the catalytic rates originating from conformational fluctuations. Although these experiments are still in their infancy, they may be able to relate the topography of the energy landscape to the biological function and regulation of enzymes. This review summarizes some of the experimental techniques and data-analysis methods that have been used to study individual enzyme molecules in search of a deeper understanding of their kinetics.

Site-specific immobilization of genetically engineered variants of Candida antarctica lipase B.

The immobilization of proteins on solid surfaces has been a topicof intensive researc h for many years. Numerous methods have been developed to immobilize proteins for bioseparation, biosensors, diagnostictests and single-molec ule experiments. [1] With the growing need for miniaturization and parallelization, new methods are needed for the immobilization of proteins with high functional density and specificity. [2, 3] Most of the methods used so far do not allow the covalent immobilization of a protein at a well-defined position. In this study, cysteines, introduced by genetic engineering, have been used for site-specific immobilization of a model enzyme to a glass surface by means of a heterobifunctional poly(ethylene glycol) (PEG) spacer. [4] While PEG is used for a broad range of applications to render surfaces protein resistant, [5–7] only a few reports describe its use as a linker for attaching proteins to surfaces. [8–13] However, the use of a heterobifunctional PEG spacer would provide a protein-resistant surface displaying reactive groups for the covalent attachment of proteins in a controlled manner. In addition, the coupling of PEG to proteins has been shown to increase the stability of the protein and maintain it in an active conformation. [14] To evaluate the usefulness of this concept, Candida antarctica lipase B (CalB; EC 3.1.1.3) was used as a model enzyme. [15] CalB is an industrially important lipase that is used for various applications in bioorganic synthesis. [16] It is a 35 kDa monomer with three disulfide bridges. CalB has been immobilized on surfaces for various applications, but these methods are mainly based on the adsorption of hydrophobicor c harged amino acids to supports. [17] Site-specific immobilization of lipases would be advantageous because it has been shown that the properties of lipases depend on their orientation on a surface. [18] In this study, mutants of CalB have been prepared that display a free cysteine at a defined position on the molecule

Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen.

The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains. The crystals have a solvent content of 77% (v/v) and form 70 A-wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.

Fluorescence-based analysis of enzymes at the single-molecule level.

Enzymes, and proteins in general, consist of a dynamic ensemble of different conformations, which fluctuate around an average structure. Single‐molecule experiments are a powerful tool to obtain information about these conformations and their contributions to the catalytic reaction. In contrast to classical ensemble measurements, which average over the whole population, singlemolecule experiments are able to detect conformational heterogeneities, to identify transient or rare conformations, to follow the time series of conformational changes and to reveal parallel reaction pathways. A number of single‐molecule studies with enzymes have proven this potential showing that the activity of individual enzymes varies between different molecules and that the catalytic rate constants fluctuate over time. From a practical point of view this review focuses on fluorescence‐based methods that have been used to study enzymes at the single‐molecule level. Since the first proof‐of‐principle experiments a wide range of different methods have been developed over the last 10 years and the methodology now needs to be applied to answer questions of biological relevance, for example about conformational changes induced by allosteric effectors or mutations.