Project description:Mechanistic information about how gaseous ions are formed from charged droplets has been difficult to establish because direct observation of nanodrops in a size range relevant to gaseous macromolecular ion formation by optical or traditional mass spectrometry methods is challenging owing to their small size and heterogeneity. Here, the mass and charge of individual aqueous nanodrops between 1-10 MDa (15-32 nm diameter) with ∼50-300 charges are dynamically monitored for 1 s using charge detection mass spectrometry. Discrete losses of minimally solvated singly charged ions occur, marking the first direct observation of ion emission from aqueous nanodrops in late stages of droplet evaporation relevant to macromolecular ion formation in native mass spectrometry. Nanodrop charge depends on the identity of constituent ions, with pure water nanodrops charged slightly above the Rayleigh limit and aqueous solutions containing alkali metal ions charged progressively below the Rayleigh limit with increasing cation size. MS2 capsid ions (∼3.5 MDa; ∼27 nm diameter) are more highly charged from aqueous ammonium acetate than from its biochemically preferred, 100 mM NaCl/10 mM Na phosphate solution, consistent with ion emission reducing the nanodrop and resulting capsid charge. The extent of charging indicates that the capsid partially collapses inside the nanodrops prior to the charging and formation of the dehydrated gaseous ions. These results demonstrate that ion emission can affect macromolecular charging and that conformational changes to macromolecular structure can occur in nanodrops prior to the formation of naked gaseous ions.
Project description:Second-order nonlinear spectroscopy has proven to be a powerful tool in elucidating key chemical and structural characteristics at a variety of interfaces. However, the presence of interfacial potentials may lead to complications regarding the interpretation of second harmonic and vibrational sum frequency generation responses from charged interfaces due to mixing of absorptive and dispersive contributions. Here, we examine by means of mathematical modeling how this interaction influences second-order spectral lineshapes. We discuss our findings in the context of reported nonlinear optical spectra obtained from charged water/air and solid/liquid interfaces and demonstrate the importance of accounting for the interfacial potential-dependent χ (3) term in interpreting lineshapes when seeking molecular information from charged interfaces using second-order spectroscopy.
Project description:Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximately 100 micros and distances greater than approximately 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-micros decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ("D(phys)"). D(phys) had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the "speed limit" of folding. D(phys) exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.
Project description:The distribution of ions and charge at solid-water interfaces plays an essential role in a wide range of processes in biology, geology and technology. While theoretical models of the solid-electrolyte interface date back to the early 20th century, a detailed picture of the structure of the electric double layer has remained elusive, largely because of experimental techniques have not allowed direct observation of the behaviour of ions, i.e. with subnanometer resolution. We have made use of recent advances in high-resolution Atomic Force Microscopy to reveal, with atomic level precision, the ordered adsorption of the mono- and divalent ions that are common in natural environments to heterogeneous gibbsite/silica surfaces in contact with aqueous electrolytes. Complemented by density functional theory, our experiments produce a detailed picture of the formation of surface phases by templated adsorption of cations, anions and water, stabilized by hydrogen bonding.
Project description:In this study, we describe a simple method to mark specific sequences in double-stranded DNA molecules. For the marking, we used two specifically designed oligonucleotides, one of which is complementary to the sequence to be marked and the other, serving as a splint, to make the marking stable and detectable by subsequent various analytical means. In the presence of the two deoxyoligonucleotides, whereas RecA protein-mediated reaction converts the sequence to be marked to a regional triple-stranded structure with the complementary (probing) oligonucleotide, DNA ligase transforms it to a stable multi- (possibly quintuple) stranded structure with the splint oligonucleotide. The whole marking process is simple and completed in a single reaction mixture. Because RecA protein makes the marking to proceed with high fidelity, we were able to mark (detect) SNPs in complex genomes like human's. Furthermore, the structure of the marked sequence is stable and quite distinct enough to be readily detectable by biochemical means or direct observation by scanning probe microscopy.
Project description:Silicon nanowires inspire since decades a great interest for their fundamental scientific importance and their potential in new technologies. When decorated with organic molecules they form hybrid composites with applications in various fields, from sensors to life science. Specifically the diethyl 1-propylphosphonate/Si combination is considered as a promising alternative to the conventional semiconductor n-type doping methods, thanks to its solution-based processing, which is damage-free and intrinsically conformal. For these characteristics, it is a valid doping process for patterned materials and nanostructures such as the nanowires. Our joined experimental and theoretical study provides insights at atomistic level on the molecular activation, grafting and self-assembling mechanisms during the deposition process. For the first time to the best of our knowledge, by using scanning transmission electron microscopy the direct visualization of the single molecules arranged over the Si nanowire surface is reported. The results demonstrate that the molecules undergo to a sequential decomposition and self-assembling mechanism, finally forming a chemical bond with the silicon atoms. The ability to prepare well-defined molecule decorated Si nanowires opens up new opportunities for fundamental studies and nanodevice applications in diverse fields like physics, chemistry, engineering and life sciences.
Project description:Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (?12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.
Project description:Amyloid aggregates are associated with a range of human neurodegenerative disorders, and it has been shown that neurotoxicity is dependent on aggregate size. Combining molecular simulation with analytical theory, a predictive model is proposed for the adsorption of amyloid aggregates onto oppositely charged surfaces, where the interaction is governed by an interplay between electrostatic attraction and entropic repulsion. Predictions are experimentally validated against quartz crystal microbalance-dissipation experiments of amyloid beta peptides and fragmented fibrils in the presence of a supported lipid bilayer. Assuming amyloids as rigid, elongated particles, we observe nonmonotonic trends for the extent of adsorption with respect to aggregate size and preferential adsorption of smaller aggregates over larger ones. Our findings describe a general phenomenon with implications for stiff polyions and rodlike particles that are electrostatically attracted to a surface.
Project description:Despite the importance of the hydrogen ion in a wide range of biological, chemical, and physical processes, its molecular structure in solution remains lively debated. Progress has been primarily hampered by the extreme diffuse nature of the vibrational signatures of hydrated protons in bulk solution. Using the inherently surface-specific vibrational sum frequency spectroscopy technique, we show that at selected negatively charged interfaces, a resolved spectral feature directly linked to the H3O+ core in an Eigen-like species can be readily identified in a biologically compatible pH range. Centered at ~2540 cm-1, the band is seen to shift to ~1875 cm-1 when forming D3O+ upon isotopic substitution. The results offer the possibility of tracking and understanding from a molecular perspective the behavior of hydrated protons at charged interfaces.
Project description:A surface sensitive second order nonlinear optical technique, sum frequency generation vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy. Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, for example, air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation, and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities.