José Juan Colás

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 multi-angle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single sub-micron 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 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.

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotech-nological applications, including viral inactivation and protein extraction. Although the ensemble thermo-dynamics 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 multi-angle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single sub-micron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on in-tact 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 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.

The formation of the mollusk shell requires the participation of proteins, many of which may be interactive with one an-other. 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 AP24 glycosylation and primary sequence had on AP24-AP7 binding. We find that both the glycosylated and non-glycosylated variants of AP24 bound to AP7 but with different quantities, kinetics, and internal rearrangements. Moreover, the binding of AP7 with non-glycosylated 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 nacre matrix formation.

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.

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.

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.