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Multivalent ligand-receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5(Van) n ( n = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5(Van) 4 bound most tightly with a K D of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5(Van) 1 or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5(Van) n and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at n = 4 greater than 96 pN at n = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates.

We report the application of recently developed microscopic models to estimate the apparent kinetic and thermodynamic parameters in a single molecule force spectroscopy study of the carbonic anhydrase enzyme and a complementary sulfonamide inhibitor. The most probable rupture force for the enzyme-inhibitor interaction shows a nonlinear dependency on the log-loading rate. Estimates for the kinetic and thermodynamic parameters were obtained by fitting the nonlinear dependency to linear cubic potential and cusp potential models and compared to the standard Bell-Evans model. The reliability of the estimated parameters was verified by modeling the experimental rupture force distributions by the theoretically predicted distributions at rupture. We also report that linkers that are attached to the enzyme and inhibitor show appreciable effects on the apparent kinetic and thermodynamic parameters.

In this communication, we report on the interaction landscape of an active site-specific enzyme-inhibitor complex by single-molecule force spectroscopy. Electrostatic immobilization was employed to orient a carbonic anhydrase enzyme on a positively charged surface so its active site is pointing upward. This approach to immobilization effectively increases the number of specific interactions measured between the zinc ion of the active site on carbonic anhydrase and a sulfonamide inhibitor tethered to an atomic force microscope (AFM) probe. Further, it reduces the time required for data collection and thereby minimizes the possible mechanical damage to the probe and contamination of the enzyme surface. The rupture force measured at various loading rates is interpreted in terms of a single energy barrier for the carbonic anhydrase enzyme-sulfonamide inhibitor complex from which the kinetic and thermodynamic parameters were estimated on the basis of microscopic models and were compared to the Bell-Evans model. The dissociation rate for the enzyme-inhibitor complex was found to be significantly faster (~35 times) than the natural spontaneous dissociation rate.

We report that varying the contact force in force spectroscopy results in a significant shift in DNA unbinding forces, measured from short oligonucleotides using a PicoForce microscope. The contact force between a 30-mer complementary DNA-coated probe and surface was varied from 100 pN to 10 nN, resulting in a significant shift in the most abundant unbinding force measured between the duplex. When contact forces were set at 200 pN or less, which is generally considered to be a low contact force region for biomolecular force spectroscopy studies, the shift in DNA unbinding forces was significant with changes in contact force. The effect of the salt concentration on the DNA unbinding forces was also examined for a range of salt concentrations from 5 to 500 mM because the presence of salt ions is necessary to facilitate the hybridization process. Although an increase in salt concentration resulted in the facilitation of DNA multiple binding events during force spectroscopy measurements, no significant shift in unbinding forces was observed. Our experiment demonstrates that the wide variation in DNA unbinding forces reported in the literature (50-600 pN) for short oligonucleotides can be accounted for by the different contact forces used and shows little or no effect of the salt concentration used in those studies. Furthermore, this study demonstrates the importance of reporting contact forces in force spectroscopy measurements for quantitative comparisons between different biomolecular systems, especially for noncovalent-type interactions.

In this letter, we show that electrostatic immobilization provides a simple but effective approach for the immobilization and orientation of carbonic anhydrase onto charged surfaces. The enzyme is oriented differently on oppositely charged surfaces, with the majority of active sites facing upward on a positively charged surface and downward on a negatively charged surface. An array of negatively charged microscale surface patterns within a positively charged background was prepared by microcontact printing and used as the substrate to immobilize the enzymes. This enabled the probing of the enzyme orientations on the two differently charged surface regions by force spectroscopy with the same atomic force microscopy (AFM) probe modified with a thiolated sulfonamide inhibitor. The unbinding forces between the inhibitor tip and the enzyme immobilized on the two differently charged surfaces were measured. Two control experiments, blocking of the enzyme active site with a competitive inhibitor and removal of the zinc ion from the enzyme catalytic center, were employed to distinguish between specific and nonspecific interactions and to further verify the differences in enzyme orientation. Autocorrelation analysis of the force histograms was carried out to evaluate the specific single enzyme-inhibitor interaction force.