Measuring the p<i>K</i>/p<i>I</i> of Biomolecules Using X‑ray Photoelectron Spectroscopy

Dissociation constants of GG–X–GG and X₅ peptides (X = G, D, H, or K), and bovine albumin (BSA) and fibronectin (FN) were measured by X-ray photoelectron spectroscopy (XPS) in ultrahigh vacuum at room temperature. The biomolecules were deposited on Au substrates by drying 2.0 μL drops of 1.0 μg μL^–1 stock solutions in 100 mM sodium phosphate buffers (pH 1–12) at room temperature. Because of the ∼+1.3 eV shift in binding energy (BE) of protonated amines, p<i>K</i> values of basic amino acids were calculated by plotting the fraction of protonated amines as a function of solution pH. Similarly, the BE of carboxyl groups shifted ∼−1.3 eV upon deprotonation. While C 1s spectra were convoluted by the multiple chemical states of carbon present in the samples, the ratio of the C 1s components centered at BE = 289.0 ± 0.4 and BE = 287.9 ± 0.3 proved to reliably assess deprotonation of carboxyl groups. The p<i>K</i> values for the Asp (3.1 and 2.4), His (6.7), and Lys (11.3 and 10.6) peptides, and the p<i>I</i> of BSA (4.8) and FN (5.7), were consistent with published values; thus, these methods could potentially be used to determine the dissociation constants of surface-bound biomolecules

Self-Assembly of Protein Nanofibrils Orchestrates Calcite Step Movement through Selective Nonchiral Interactions

The recognition of atomically distinct surface features by adsorbed biomolecules is central to the formation of surface-templated peptide or protein nanostructures. On mineral surfaces such as calcite, biomolecular recognition of, and self-assembly on, distinct atomic kinks and steps could additionally orchestrate changes to the overall shape and symmetry of a bulk crystal. In this work, we show through <i>in situ</i> atomic force microscopy (AFM) experiments that an acidic 20 kDa cement protein from the barnacle <i>Megabalanus rosa</i> (MRCP20) binds specifically to step edge atoms on {101̅4} calcite surfaces, remains bound and further assembles over time to form one-dimensional nanofibrils. Protein nanofibrils are continuous and organized at the nanoscale, exhibiting striations with a period of ca. 45 nm. These fibrils, templated by surface steps of a preferred geometry, in turn selectively dissolve underlying calcite features displaying the same atomic arrangement. To demonstrate this, we expose the protein solution to bare and fibril-associated rhombohedral etch pits to reveal that nanofibrils accelerate only the movement of fibril-forming steps when compared to undecorated steps exposed to the same solution conditions. Calcite mineralized in the presence of MRCP20 results in asymmetric crystals defined by frustrated faces with shared mirror symmetry, suggesting a similar step-selective behavior by MRCP20 in crystal growth. As shown here, selective surface interactions with step edge atoms lead to a cooperative regime of calcite modification, where templated long-range protein nanostructures shape crystals

Layer-by-Layer Assembly of Heterogeneous Modular Nanocomposites

Layer-by-layer (LbL) assembly of nanoparticles and polyelectrolyte multilayers into alternating nonrepetitive strata (modules) demonstrates an important advance in heterogeneous nanocomposites from random or repetitive distributions of nanoparticles to a versatile modular design. The morphology and composition for each module are determined by the LbL assembly conditions, as confirmed by cross-section transmission electron microscopy (TEM) and by X-ray photoelectron spectroscopy (XPS). Thickness and spacing of the modules are maintained in the 5–50 nm range relevant for nanoscale proximal interactions

Spanning Strong to Weak Normal Mode Coupling between Vibrational and Fabry–Pérot Cavity Modes through Tuning of Vibrational Absorption Strength

Designing coupled vibrational-cavity polariton systems to modify chemical reaction rates and paths requires an understanding of how this coupling depends on system parameters (i.e., absorber strength, modal distribution, and vibrational absorber and cavity line widths). Here, we evaluate the impact of absorption coefficient and cavity design on normal mode coupling between a Fabry–Pérot cavity and a molecular vibration. For a vibrational band of urethane in a polymer matrix, the coupling strength increases with its concentration so that the system spans the weak and strong coupling regimes. The experimentally determined Rabi splitting values are in excellent agreement with an analytical expression derived for classical coupled oscillators that includes no fitting parameters. Also, the cavity mode profile is altered through choice of mirror type, with metal mirrors resulting in stronger confinement and thus coupling, while dielectric stack mirrors provide higher transmission for a given cavity quality factor and decreased coupling due to greater mode penetration into the dielectric mirror. In addition to polymers, the cavities can couple to molecular vibrational bands of dissolved species in solution, which greatly expands the range of systems that can be explored. Finally, longer path length cavities are used to demonstrate the path length independence of the coupling strength. The ability to adjust the cavity line width, through the use of higher order modes, represents a route to match the cavity dephasing time to that of the molecular vibration and may be applied to a range of molecular systems. Understanding the roles of cavity design and validating empirical and analytical descriptions of absorber properties on coupling strength will facilitate application of these strong coupling effects to enable currently unreachable chemistries

Growth and development of the barnacle <i>Amphibalanus amphitrite</i>: time and spatially resolved structure and chemistry of the base plate

<div><p>The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, <i>Amphibalanus</i> (= <i>Balanus</i>) <i>amphitrite</i> were resolved <i>in situ</i> and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide <i>ex situ</i> material identification of regions imaged by confocal microscopy. <i>In situ</i> and <i>ex situ</i> analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.</p></div

Imaging Active Surface Processes in Barnacle Adhesive Interfaces

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor <i>Amphibalanus (=Balanus) amphitrite</i> barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces

Imaging Active Surface Processes in Barnacle Adhesive Interfaces

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor <i>Amphibalanus (=Balanus) amphitrite</i> barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces

Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady’s reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43