The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate.

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotechnological applications, including viral inactivation and protein extraction. Although the ensemble thermodynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multiangle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single submicron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on intact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization, and we expect that the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.

A Complicated Relationship: Glycosylation, Ca(II), and Primary Sequence Affect the Interactions and Kinetics between Two Model Mollusk Shell Intracrystalline Nacre Proteins.

The formation of the mollusk shell requires the participation of proteins, many of which may be interactive with one another. We examined a model protein pair system from the mollusk Haliotis rufescens, wherein we probed the interactions between recombinant forms of two major nacre layer proteins, AP7, and the glycoprotein, AP24. Here, the focus was on the impact that the AP24 glycosylation and primary sequence had on AP24-AP7 binding. We find that both the glycosylated and nonglycosylated variants of AP24 bound to AP7 but with different quantities, kinetics, and internal rearrangements. Moreover, the binding of AP7 with nonglycosylated and glycosylated AP24 was found to be Ca(II)-dependent and -independent, respectively. Yet both variants of AP24 combine with AP7 to form hybrid hydrogel particles that are similar in their physical properties. Thus, AP7 and AP24 protein sequences are interactive and form hydrogels, but the interactions are tuned by glycosylation and Ca(II). These features may have an impact on the nacre matrix formation.

Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

Quantifying single-cell secretion in real time using resonant hyperspectral imaging.

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.

Dual-Mode Electro-Optical Techniques for Biosensing Applications: A Review.

The monitoring of biomolecular interactions is a key requirement for the study of complex biological processes and the diagnosis of disease. Technologies that are capable of providing label-free, real-time insight into these interactions are of great value for the scientific and clinical communities. Greater understanding of biomolecular interactions alongside increased detection accuracy can be achieved using technology that can provide parallel information about multiple parameters of a single biomolecular process. For example, electro-optical techniques combine optical and electrochemical information to provide more accurate and detailed measurements that provide unique insights into molecular structure and function. Here, we present a comparison of the main methods for electro-optical biosensing, namely, electrochemical surface plasmon resonance (EC-SPR), electrochemical optical waveguide lightmode spectroscopy (EC-OWLS), and the recently reported silicon-based electrophotonic approach. The comparison considers different application spaces, such as the detection of low concentrations of biomolecules, integration, the tailoring of light-matter interaction for the understanding of biomolecular processes, and 2D imaging of biointeractions on a surface.

Functional Prioritization and Hydrogel Regulation Phenomena Created by a Combinatorial Pearl-Associated Two-Protein Biomineralization Model System.

In the nacre or aragonitic layer of an oyster pearl, there exists a 12-member proteome that regulates both the early stages of nucleation and nanoscale-to-mesoscale assembly of nacre tablets and calcitic crystals from mineral nanoparticle precursors. Several approaches to understanding protein-associated mechanisms of pearl nacre formation have been developed, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights, we have created a proportionally defined combinatorial model consisting of two pearl nacre-associated proteins, PFMG1 and PFMG2 (shell oyster pearl nacre, Pinctada fucata) whose individual in vitro mineralization functionalities are distinct from one another. Using scanning electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that at 1:1 molar ratios, rPFMG2 and rPFMG1 co-aggregate in specific molecular ratios to form hybrid hydrogels that affect both the early and later stages of in vitro calcium carbonate nucleation. Within these hybrid hydrogels, rPFMG2 plays a role in defining protein co-aggregation and hydrogel dimension, whereas rPFMG1 defines participation in nonclassical nucleation processes; both proteins exhibit synergy with regard to surface and subsurface modifications to existing crystals. The interactions between both proteins are enhanced by Ca(II) ions and may involve Ca(II)-induced conformational events within the EF-hand rPFMG1 protein, as well as putative interactions between the EF-hand domain of rPFMG1 and the calponin-like domain of rPFMG2. Thus, the pearl-associated PFMG1 and PFMG2 proteins interact and exhibit mineralization functionalities in specific ways, which may be relevant for pearl formation.

The electrophotonic silicon biosensor.

The emergence of personalized and stratified medicine requires label-free, low-cost diagnostic technology capable of monitoring multiple disease biomarkers in parallel. Silicon photonic biosensors combine high-sensitivity analysis with scalable, low-cost manufacturing, but they tend to measure only a single biomarker and provide no information about their (bio)chemical activity. Here we introduce an electrochemical silicon photonic sensor capable of highly sensitive and multiparameter profiling of biomarkers. Our electrophotonic technology consists of microring resonators optimally n-doped to support high Q resonances alongside electrochemical processes in situ. The inclusion of electrochemical control enables site-selective immobilization of different biomolecules on individual microrings within a sensor array. The combination of photonic and electrochemical characterization also provides additional quantitative information and unique insight into chemical reactivity that is unavailable with photonic detection alone. By exploiting both the photonic and the electrical properties of silicon, the sensor opens new modalities for sensing on the microscale.