Looking inside molecular bonds at biological interfaces with dynamic force spectroscopy

Weak non-covalent interactions between large molecules govern interfacial structure and adhesion in biology. Because of thermal activation, these bonds have modest lifetimes and bond lifetimes are progressively shortened under application of external force. Theory predicts that bond survival time depends on how fast the force is applied and the expected survival time specifies the most likely breakage force (strength) at a given loading rate (force/time). Plotted as a function of log(e) (loading rate), the dynamic spectrum of bond strength provides an image of the prominent barriers traversed in the energy landscape along the unbinding pathway, which establishes a direct link between measurements of bond force and molecular-scale chemistry. Experimentally, the challenge is to measure bond strength over several orders of magnitude in loading rate. With a recently designed probe technique, we have measured strengths of single receptor-ligand bonds and receptor-membrane anchoring over an enormous range of loading rates from 10(-1) pN/s to 10(5) pN/s, which reveals an inner view of the complexity of these interactions.     

FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation

The bacterial adhesive protein, FimH, is the most common adhesin of Escherichia coli and mediates weak adhesion at low flow but strong adhesion at high flow. There is evidence that this occurs because FimH forms catch bonds, defined as bonds that are strengthened by tensile mechanical force. Here, we applied force to single isolated FimH bonds with an atomic force microscope in order to test this directly. If force was loaded slowly, most of the bonds broke up at low force (<60 piconewtons of rupture force). However, when force was loaded rapidly, all bonds survived until much higher force (140-180 piconewtons of rupture force), behavior that indicates a catch bond. Structural mutations or pretreatment with a monoclonal antibody, both of which allosterically stabilize a high affinity conformation of FimH, cause all bonds to survive until high forces regardless of the rate at which force is applied. Pretreatment of FimH bonds with intermediate force has the same strengthening effect on the bonds. This demonstrates that FimH forms catch bonds and that tensile force induces an allosteric switch to the high affinity, strong binding conformation of the adhesin. The catch bond behavior of FimH, the amount of force needed to regulate FimH, and the allosteric mechanism all provide insight into how bacteria bind and form biofilms in fluid flow. Additionally, these observations may provide a means for designing antiadhesive mechanisms.     

Energy of hydrogen bonds probed by the adhesion of functionalized lipid layers

It is now well admitted that hydrophobic interactions and hydrogen bonds are the main forces driving protein folding and stability. However, because of the complex structure of a protein, it is still difficult to separate the different energetic contributions and have a reliable estimate of the hydrogen bond part. This energy can be quantified on simpler systems such as surfaces bearing hydrogen-bonding groups. Using the surface force apparatus, we have directly measured the interaction energy between monolayers of lipids whose headgroups can establish hydrogen bonds in water: nitrilotriacetate, adenosine, thymidine, and methylated thymidine lipids. From the adhesion energy between the surfaces, we have deduced the energy of a single hydrogen bond in water. We found in each case an energy of 0.5 kcal/mol. This result is in good agreement with recent experimental and theoretical studies made on protein systems showing that intramolecular hydrogen bonds make a positive contribution to protein stabilization.     

Hydrogen bonds in galactopyranoside and glucopyranoside: a density functional theory study

Density functional theory calculations on two glycosides, namely, n-octyl-β-D-glucopyranoside (C₈O-β-Glc) and n-octyl-β-D-galactopyranoside (C₈O-β-Gal) were performed for geometry optimization at the B3LYP/6-31G level. Both molecules are stereoisomers (epimers) differing only in the orientation of the hydroxyl group at the C4 position. Thus it is interesting to investigate electronically the effect of the direction (axial/equatorial) of the hydroxyl group at the C4 position. The structure parameters of X-H???Y intramolecular hydrogen bonds were analyzed, while the nature of these bonds and the intramolecular interactions were considered using the atoms in molecules (AIM) approach. Natural bond orbital analysis (NBO) was used to determine bond orders, charge and lone pair electrons on each atom and effective non-bonding interactions. We have also reported electronic energy and dipole moment in gas and solution phases. Further, the electronic properties such as the highest occupied molecular orbital, lowest unoccupied molecular orbital, ionization energy, electron affinity, electronic chemical potential, chemical hardness, softness and electrophilicity index, are also presented here for both C₈O-β-Glc and C₈O-β-Gal. These results show that, while C₈O-β-Glc possess– only one hydrogen bond, C₈O-β-Gal has two intramolecular hydrogen bonds, which further confirms the anomalous stability of the latter in self-assembly phenomena.     

No energy storage in chemical bonds

References to certain biological molecules as energy sources frequently lead to the mistaken idea that energy is released when bonds are broken in these molecules. The misconception is reinforced by the widespread use of the term ‘high-energy bond’.

A group of university science majors were asked to give a molecular interpretation of the statement, ‘fats supply energy to the body’. Their responses clearly reflect the confusion of bond-energy with the energy associated with a chemical reaction.

It is suggested that energy changes in biological processes should rest on the following ideas:

• Single interactions in chemical systems involve formation or breaking of bonds.

• Formation of a bond is always accompanied by a release of energy, and breaking a bond always requires energy.

• Energy changes in chemical processes are the net result of the breaking and formation of bonds.

In the interests of a sound understanding of energy transformations, the high-energy bond idea should be treated cautiously. The Nuffield A-level Chemistry treatment of ATP in intermediary metabolism has even entirely avoided the concept of a high-energy bond.     

C-H...O hydrogen bonds in the nuclear receptor RARgamma--a potential tool for drug selectivity

Hydrogen bonds between polarized atoms play a crucial role in protein interactions and are often used in drug design, which usually neglects the potential of C-H...O hydrogen bonds. The 1.4 A resolution crystal structure of the ligand binding domain of the retinoic acid receptor RARgamma complexed with the retinoid SR11254 reveals several types of C-H...O hydrogen bonds. A striking example is the hydroxyl group of the ligand that acts as an H bond donor and acceptor, leading to a synergy between classical and C-H...O hydrogen bonds. This interaction introduces both specificity and affinity within the hydrophobic ligand pocket. The similarity of intraprotein and protein-ligand C-H...O interactions suggests that such bonds should be considered in rational drug design approaches.     

Strength of multiple parallel biological bonds

Multivalent interactions play a critical role in a variety of biological processes on both molecular and cellular levels. We have used molecular force spectroscopy to investigate the strength of multiple parallel peptide-antibody bonds using a system that allowed us to determine the rupture forces and the number of ruptured bonds independently. In our experiments the interacting molecules were attached to the surfaces of the probe and sample of the atomic force microscope with flexible polymer tethers, and the unique mechanical signatures of the tethers determined the number of ruptured bonds. We show that the rupture forces increase with the number of interacting molecules and that the measured forces obey the predictions of a Markovian model for the strength of multiple parallel bonds. We also discuss the implications of our results to the interpretation of force spectroscopy measurements in multiple bond systems.     

Dynamic strength of molecular adhesion bonds.

In biology, molecular linkages at, within, and beneath cell interfaces arise mainly from weak noncovalent interactions. These bonds will fail under any level of pulling force if held for sufficient time. Thus, when tested with ultrasensitive force probes, we expect cohesive material strength and strength of adhesion at interfaces to be time- and loading rate-dependent properties. To examine what can be learned from measurements of bond strength, we have extended Kramers' theory for reaction kinetics in liquids to bond dissociation under force and tested the predictions by smart Monte Carlo (Brownian dynamics) simulations of bond rupture. By definition, bond strength is the force that produces the most frequent failure in repeated tests of breakage, i.e., the peak in the distribution of rupture forces. As verified by the simulations, theory shows that bond strength progresses through three dynamic regimes of loading rate. First, bond strength emerges at a critical rate of loading (> or = 0) at which spontaneous dissociation is just frequent enough to keep the distribution peak at zero force. In the slow-loading regime immediately above the critical rate, strength grows as a weak power of loading rate and reflects initial coupling of force to the bonding potential. At higher rates, there is crossover to a fast regime in which strength continues to increase as the logarithm of the loading rate over many decades independent of the type of attraction. Finally, at ultrafast loading rates approaching the domain of molecular dynamics simulations, the bonding potential is quickly overwhelmed by the rapidly increasing force, so that only naked frictional drag on the structure remains to retard separation. Hence, to expose the energy landscape that governs bond strength, molecular adhesion forces must be examined over an enormous span of time scales. However, a significant gap exists between the time domain of force measurements in the laboratory and the extremely fast scale of molecular motions. Using results from a simulation of biotin-avidin bonds (Izrailev, S., S. Stepaniants, M. Balsera, Y. Oono, and K. Schulten. 1997. Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys. J., this issue), we describe how Brownian dynamics can help bridge the gap between molecular dynamics and probe tests.     

Competition between hydrogen bonds and halogen bonds in complexes of formamidine and hypohalous acids

Quantum chemical calculations have been per-formed for the complexes of formamidine (FA) and hypohalous acid (HOX, X = F, Cl, Br, I) to study their structures, properties, and competition of hydrogen bonds with halogen bonds. Two types of complexes are formed mainly through a hydrogen bond and a halogen bond, respectively, and the cyclic structure is more stable. For the F, Cl, and Br complexes, the hydrogen-bonded one is more stable than the halogen-bonded one, while the halogen-bonded structure is favorable for the I complexes. The associated H-O and X-O bonds are elongated and exhibit a red shift, whereas the distant ones are contracted and display a blue shift. The strength of hydrogen and halogen bonds is affected by F and Li substitutents and it was found that the latter tends to smooth differences in the strength of both types of interactions. The structures, properties, and interaction nature in these complexes have been understood with natural bond orbital (NBO) and atoms in molecules (AIM) theories.     

NMR evaluation of adipocyte fatty acid binding protein (aP2) with R- and S-ibuprofen

We have examined global chemical shift perturbations for aP2 ligand complexes and compared these with amide temperature coefficients. Hydrogen bond potential was monitored by amide chemical shift's temperature coefficient. Based on this information, we propose that the binding energy contribution can be spread out to multiple distant residues. For aP2, the ability of the receptor protein to change its hydrogen bond interactions in the beta-strands to accommodate different ligand scaffolds seems to make this receptor difficult for structure based drug design. While stabilization energy differential on hydrogen bonds is likely to be small for individual residues, the accumulative effect on multiple hydrogen bonds may have a dramatic impact on ligand affinity.     

Elasticity-associated rebinding rate of molecular bonds between soft elastic media

A quantitative understanding of how cells interact with their extracellular matrix via molecular bonds is fundamental for many important processes in cell biology and engineering. In these interactions, the deformability of cells and matrix are usually comparable with that of the bonds, making their rebinding events globally coupled with the deformation states of whole systems. Unfortunately, this important principle is not realized or adopted in most conventional theoretical models for analyzing cellular adhesions. In this study, we considered a new theoretical model of a cluster of ligand-receptor bonds between two soft elastic bodies, in which the rebinding rates of ligands to receptors are described, by considering the deformation of the overall system under the influence of bond distributions. On the basis of theory of continuum and statistical mechanics, we obtained an elasticity-associated rebinding rate of open bonds in a closed analytical form that highly depends on the binding states and distributions of all other bonds as well as on the overall deformation energy stored in the elastic bodies and all closed bonds. On the basis of this elasticity-associated rebinding rate and by performing Monte Carlo simulations, we uncovered new mechanisms underlying the adhesion stability of molecular-bond clusters associated with deformable elastic bodies. Moreover, we revealed that the rebinding processes of molecular bonds is not only dependent on interfacial separation but is related to overall energy. This newly proposed rebinding rate may substantially improve our understanding of how cells adapt to their microenvironments by adjusting their mechanical properties through cytoskeleton remodeling.     

C‐H…O hydrogen bonds in FK506‐binding protein–ligand interactions

Hydrogen bonds are important interaction forces observed in protein structures. They can be classified as stronger or weaker depending on their energy, thereby reflecting on the type of donor. The contribution of weak hydrogen bonds is deemed as an important factor toward structure stability along with the stronger bonds. One such bond, the C‐H…O type hydrogen bond, is shown to make a contribution in maintaining three dimensional structures of proteins. Apart from their presence within protein structures, the role of these bonds in protein–ligand interactions is also noteworthy. In this study, we present a statistical analysis on the presence of C‐H…O hydrogen bonds observed between FKBPs and their cognate ligands. The FK506‐binding proteins (FKBPs) carry peptidyl cis–trans isomerase activity apart from the immunosuppressive property by binding to the immunosuppressive drugs FK506 or rapamycin. Because the active site of FKBPs is lined up by many hydrophobic residues, we speculated that the prevalence of C‐H…O hydrogen bonds will be considerable. In a total of 25 structures analyzed, a higher frequency of C‐H…O hydrogen bonds is observed in comparison with the stronger hydrogen bonds. These C‐H…O hydrogen bonds are dominated by a highly conserved donor, the C^α/β of Val55 and an acceptor, the backbone oxygen of Glu54. Both these residues are positioned in the β4‐α1 loop, whereas the other residues Tyr26, Phe36 and Phe99 with higher frequencies are lined up at the opposite face of the active site. These preferences could be implicated in FKBP pharmacophore models toward enhancing the ligand affinity. This study could be a prelude to studying other proteins with hydrophobic pockets to gain better insights into ligand recognition. Copyright © 2013 John Wiley & Sons, Ltd.     

Hydrogen bonds in rubredoxins from mesophilic and hyperthermophilic organisms

The extent and strength of the hydrogen bond networks in rubredoxins from the hyperthermophile Pyrococcus furiosus (PfRd), and its mesophilic analogue Clostridium pasteurianum (CpRd), are examined and compared using NMR spectroscopy. NMR parameters examined in this study include through-hydrogen bond (h3)J(NC)(') scalar couplings and (1)H, (13)C, and (15)N chemical shifts, as well as covalent (1)J(NH) and (1)J(NC)(') scalar couplings. These parameters have allowed the characterization in solution of 12 hydrogen bonds in each protein. Despite a 83% sequence homology and a low RMSD for the backbone heavy atoms (0.648 A) in the crystalline state, subtle, but definite, changes have been identified in the detailed hydrogen-bonding patterns. CpRd shows an increased number of hydrogen bonds in the triple-stranded beta-sheet and an additional hydrogen bond in the multiple-turn segment including residues 14-32. On the other hand, PfRd exhibits an overall strengthening of N-H...O=C hydrogen bonds in the loops involved at the metal binding site as well as evidence for an additional NH...S(Cys) hydrogen bond involving the alanine residue 44. These data, as well as temperature dependence of the NMR parameters, suggest that the particular NMR hydrogen bond pattern found in the hyperthermophile rubredoxin leads to an increased stabilization at the metal binding pocket. It seems to result from a subtle redistribution of hydrogen-bonding interactions between the triple-stranded beta-sheet and the actual metal binding site.     

A structure-based sliding-rebinding mechanism for catch bonds

Catch bonds, whose lifetimes are prolonged by force, have been observed in selectin-ligand interactions and other systems. Several biophysical models have been proposed to explain this counterintuitive phenomenon, but none was based on the structure of the interacting molecules and the noncovalent interactions at the binding interface. Here we used molecular dynamics simulations to study changes in structure and atomic-level interactions during forced unbinding of P-selectin from P-selectin glycoprotein ligand-1. A mechanistic model for catch bonds was developed based on these observations. In the model, “catch” results from forced opening of an interdomain hinge that tilts the binding interface to allow two sides of the contact to slide against each other. Sliding promotes formation of new interactions and even rebinding to the original state, thereby slowing dissociation and prolonging bond lifetimes. Properties of this sliding-rebinding mechanism were explored using a pseudoatom representation and Monte Carlo simulations. The model has been supported by its ability to fit experimental data and can be related to previously proposed two-pathway models.     

Cooperative adhesion of ligand-receptor bonds

Cooperative (simultaneous) breakage of multiple adhesive bonds has been proposed as a mechanism for enhanced binding strength between adhesion molecules on apposing cell surfaces. In this report, we used the atomic force microscopy (AFM) to study how changes in binding affinity and separation rate of force-induced ligand-receptor dissociation affect binding cooperativity. The AFM force measurements were carried out using (strept)avidin-functionalized cantilever tips and biotinylated agarose beads under conditions where multiple (strept)avidin-biotin linkages were formed following surface contact. At slow surface separation of the AFM cantilever from the bead's surface, the (strept)avidin-biotin linkages appeared to rupture sequentially. Increasing the separation rate from 210 to 1950 nm/s led to a linear increase in the average rupture force. Moreover, force histograms revealed a quantized force distribution that shifted toward higher values with increasing separation rate. In measurements of streptavidin-iminobiotin adhesion, the force distribution also shifted toward higher values when the buffer was adjusted to a higher pH to raise the binding affinity. Together, these results demonstrate that the cooperativity of ligand-receptor bonds is significantly enhanced by increases in surface separation rate and/or binding affinity.     

Engineering disulfide bonds within an antibody

Antibodies have evolved to function in oxidative, extracellular environments. A pair of cysteines in close proximity will oxidatively react to form a disulfide bond that fixes and stabilizes the tertiary structure of a protein. Immunoglobulin G (IgG) includes several disulfide bonds, and the patterns of inter-chain disulfide bonds characterize different IgG sub-classes. Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. However, the intra-domain disulfide bond can be replaced without crucial effects on the structure and function, if the domain structure is intrinsically stable or has been stabilized by protein engineering. In previous studies, disulfide bonds were removed by amino-acid substitution indicating that Val and/or Ala (i.e. Ala–Ala, Ala–Val, Val–Ala, and Val–Ala) pairs were preferred for cysteine replacement in the Ig-fold domain. As such, these mutations may be useful for the intracellular use of antibodies. Recently, additional intra-domain disulfide bonds have been shown to stabilize Ig-fold domains and whole IgGs. In heavy chain variable or light chain variable domains, the introduction of additional disulfide bonds into the framework region did not reduce antigen-binding affinity, suggesting that generating disulfide bonds may be a method for stabilizing IgG and antibody fragments, such as the antigen-binding fragment, and single-chain and single-domain antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Allosteric disulfide bonds

Disulfide bonds have been generally considered to be either structural or catalytic. Structural bonds stabilize a protein, while catalytic bonds mediate thiol-disulfide interchange reactions in substrate proteins. There is emerging evidence for a third type of disulfide bond that can control protein function by triggering a conformational change when it breaks and/or forms. These bonds can be thought of as allosteric disulfides. To better define the properties of allosteric disulfides, we have analyzed the geometry and dihedral strain of 6874 unique disulfide bonds in 2776 X-ray structures. A total of 20 types of disulfide bonds were identified in the dataset based on the sign of the five chi angles that make up the bond. The known allosteric disulfides were all contained in 1 of the 20 groups, the -RHStaple bonds. This bond group has a high mean potential energy and narrow energy distribution, which is consistent with a functional role. We suggest that the -RHStaple configuration is a hallmark of allosteric disulfides. About 1 in 15 of all structurally determined disulfides is a potential allosteric bond.     

Specific molecular interactions by force spectroscopy: from single bonds to collective properties

Interactions involving multiple bonds occur throughout biology, and have distinct properties that are fundamentally different from those present in single bond systems. We have developed a new method to analyse the AFM force measurements in order to extract relevant information and to characterise the interactions involving from single to multiple bonds. Our study reveals a surprising behaviour in the presence of multiple bonds with a high rebinding probability: the mean binding forces increase with decreasing pulling velocity. Such behaviour is different from the force dependence on the loading rate for single bond rupture or existing models for multiple bonds rupture.     

Kinase inhibitors and the case for CH...O hydrogen bonds in protein-ligand binding

Although the hydrogen bond is known to be an important mediator of intermolecular interactions, there has yet to be an analysis of the role of CH...O hydrogen bonds in protein-ligand complexes. In this work, we present evidence for such nonstandard hydrogen bonds from a survey of aromatic ligands in 184 kinase crystal structures and 358 high-resolution structures from the Protein Data Bank. CH groups adjacent to the positively charged nitrogen of nicotinamide exhibit geometric preferences strongly suggestive of hydrogen bonding interactions, as do heterocyclic CH groups in kinase ligands, while other aromatic CH groups do not exhibit these characteristics. Ab initio calculations reveal a considerable range of CH...O hydrogen bonding potentials among different aromatic ring systems, with nicotinamide and heterocycles preferred in kinase inhibitors showing particularly favorable interactions. These results provide compelling evidence for the existence of CH...O hydrogen bonds in protein-ligand interactions, as well as information on the relative strength of various aromatic CH donors. Such knowledge will be of considerable value in protein modeling, ligand design, and structure-activity analysis.     

Competition between halogen, dihalogen and hydrogen bonds in bromo- and iodomethanol dimers

O-H…X and O-H…O H-bonds as well as C-X…X dihalogen and C-X…O halogen bonds have been investigated in halomethanol dimers (bromomethanol dimer, iodomethanol dimer, difluorobromomethanol…bromomethanol complex and difluoroiodomethanol…iodomethanol complex). Structures of all complexes were optimized at the counterpoise-corrected MP2/cc-pVTZ level and single-point energies were calculated at the CCSD(T)/aug-cc-pVTZ level. Energy decomposition for the bromomethanol dimer complex was performed using the DFT-SAPT method based on the aug-cc-pVTZ basis set. OH…O and OH…X H-bonds are systematically the strongest in all complexes investigated, with the former being the strongest bond. Halogen and dihalogen bonds, being of comparable strength, are weaker than both H-bonds but are still significant. The strongest bonds were found in the difluoroiodomethanol…iodomethanol complex, where the O-H…O H-bond exceeds 7 kcal mol^-1, and the halogen and dihalogen bonds exceed 2.5 and 2.3 kcal mol^-1, respectively. Electrostatic energy is dominant for H-bonded structures, in halogen bonded structures electrostatic and dispersion energies are comparable, and, finally, for dihalogen structures the dispersion energy is clearly dominant.