Fingerprinting polysaccharides with single-molecule atomic force microscopy

We report the use of an atomic force microscopy (AFM)-based force spectroscopy technique to identify, at the single-molecule level, the components of mixtures of polysaccharides. Previously, we showed that the elasticity of certain types of polysaccharides is governed by force-induced conformational transitions of the pyranose ring. These transitions produce atomic fingerprints in the force-extension spectrum that are characteristic of the ground-energy conformation of the pyranose ring and the type of glycosidic linkages. Using this approach we find that commercially available agarose and lambda-carrageenan contain molecules that, when stretched in an atomic force microscope, produce a force spectrum characteristic of alpha-(1-->4) d-glucans. We have identified these molecules as amylopectin or floridean starch, a storage polysaccharide in algae. Our methodology can identify individual polysaccharide molecules in solution, which is not possible by any other spectroscopic technique, and therefore is an important addition to the arsenal of analytical techniques used in carbohydrate research.     

Hydrodynamic effects in fast AFM single-molecule force measurements

Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor–ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few m/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 m/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 m/s pulling speed.Abbrevations AFM atomic force micrcoscopy - pN piconewton - BR bacteriorhodopsin - DFS dynamic force spectroscopy - Ig27 immunoglobulin 27 - If27-8 immunoglobulin 27 octameric construct - BFP biomembrane force probe     

Forced unfolding of coiled-coils in fibrinogen by single-molecule AFM

Fibrinogen is a blood plasma protein that, after activation by thrombin, assembles into fibrin fibers that form the elastic network of blood clots. We used atomic force microscopy to study the forced unfolding of engineered linear oligomers of fibrinogen, and we show that forced extension of the oligomers produces sawtooth patterns with a peak-to-peak length consistent with the independent unfolding of the coiled-coils in a cooperative two-state manner. In contrast with force plateaus seen for myosin coiled-coils that suggested rapid refolding of myosin, Monte Carlo simulations of fibrinogen unfolding confirm that fibrinogen refolding is negligible on experimental timescales. The distinct behavior of fibrinogen seems to be due to its topologically complex coiled-coils and an interaction between fibrinogen's αC-domains and its central region.     

The mechanical hierarchies of fibronectin observed with single-molecule AFM

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.     

Unequivocal single-molecule force spectroscopy of proteins by AFM using pFS vectors

Nanomechanical analysis of proteins by single-molecule force spectroscopy based on atomic force microscopy is increasingly being used to investigate the inner workings of mechanical proteins and substrate proteins of unfoldase machines as well as to gain new insight into the process of protein folding. However, such studies are hindered by a number of technical problems, including the noise of the proximal region, ambiguous single-molecule identification, as well as difficulties in protein expression/folding and full-length purification. To overcome these major drawbacks in protein nanomechanics, we designed a family of cloning/expression vectors, termed pFS (plasmid for force spectroscopy), that essentially has an unstructured region to surmount the noisy proximal region, a homomeric polyprotein marker, a carrier to mechanically protect the protein of interest (only the pFS-2 version) that also acts as a reporter, and two purification tags. pFS-2 enables the unambiguous analysis of proteins with low mechanical stability or/and complex force spectra, such as the increasingly abundant class of intrinsically disordered proteins, which are hard to characterize by traditional bulk techniques and have important biological and clinical implications. The advantages, applications, and potential of this ready-to-go system are illustrated through the analysis of representative proteins.     

Molecular docking studies on DMDP derivatives as human DHFR inhibitors

Molecular docking is routinely used for understanding drug‐receptor interaction in modern drug design. Here, we describe the docking of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). We docked 78 DMDP derivates collected from literature to DHFR and studied their specific interactions with DHFR. A new shape-based method, LigandFit, was used for docking DMDP derivatives into DHFR active sites. The result indicates that the molecular docking approach is reliable and produces a good correlation coefficient (r² = 0.499) for the 73 compounds between docking score and IC₅₀ values (Inhibitory Activity). The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme. The docked complexes provide better insights to design more potent DHFR inhibitors prior to their synthesis.     

QSAR studies on biological activity of piritrexim analogues against pc DHFR

Quantitative structure-activity relationship (QSAR) studies for 2,6-substituted 2,4-diaminopyrido[3,2-d] pyrimidine analogues of piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) are now made using topological indices. The results have shown that best models are obtained by multiparametric analysis. The predictive potential of the model is discussed on the basis of a cross-validation method.     

An RNA toolbox for single-molecule force spectroscopy studies

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.     

Protein-protein unbinding induced by force: single-molecule studies

Experiments in which two specifically interacting protein molecules are dissociated by external force have yielded new insights into mechanisms involved in cell adhesion, leukocyte rolling, regulation of integrin activity, antigen-antibody interactions and other protein-mediated reactions contingent upon molecular recognition. Another important aspect of force-induced protein-protein unbinding studies is the new information being gleaned about the thermodynamics and kinetics of bond rupture.     

Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions–diameter (D): 40–50 μm, height (H): 100 μm; H:D∼2:1). Our simulations and experiments show that the flow speed can be reduced by several orders of magnitude near the bottom of the microwells from that in the flow chamber, effectively eliminating the flow disturbance to molecules tethered in the microwells. We demonstrate a wide scope of applications of this method by measuring the force dependent DNA structural transitions in response to solution condition change, and polymerization dynamics of RecA on ssDNA/SSB-coated ssDNA/dsDNA of various tether lengths under constant forces, as well as the dynamics of vinculin binding to α-catenin at a constant force (< 5 pN) applied to the α-catenin protein.     

Laminar flow cells for single-molecule studies of DNA-protein interactions

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.     

AFM and electroanalytical studies of synthetic oligonucleotide hybridization

The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.     

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.     

Polyelectrolyte surface interface for single-molecule fluorescence studies of DNA polymerase

We report the use of polyelectrolyte multilayers in a stable robust surface chemistry for specific anchoring of DNA to glass. The nonspecific binding of fluorescently tagged nucleotides is suppressed down to the single-molecule level, and DNA polymerase is active on the anchored DNA template. This surface-chemistry platform can be used for single-molecule studies of DNA and DNA polymerase and may be more broadly applicable for other situations in which it is important to have specific biomolecular surface chemistry with extremely low nonspecific binding.     

AFM studies on gelation mechanism of xanthan gum hydrogels

The effect of annealing on xanthan gum molecules was investigated using atomic force microscopy (AFM). The values of height and width of xanthan gum molecules in AFM images are ca. 1 nm, which strongly indicates that xanthan gum molecules extended on the mica surface are in mono- or double layers. When xanthan gum aqueous solution was annealed, a network structure was observed. In contrast, a network structure was not observed for non-annealed solution. AFM images provide direct information concerning oscillational change of the network structure. It is concluded that xanthan gum molecular chains in aqueous solution aggregate and dissociate in an oscillational manner with increasing annealing time and that a homogeneous network structure was formed by annealing at 40 °C for 24 h.