Dorinel Verdes

Understanding protein adsorption phenomena at solid surfaces

Protein adsorption at solid surfaces plays a key role in many natural processes and has therefore promoted a widespread interest in many research areas. Despite considerable progress in this field there are still widely differing and even contradictive opinions on how to explain the frequently observed phenomena such as structural rearrangements, cooperative adsorption, overshooting adsorption kinetics, or protein aggregation. In this review recent achievements and new perspectives on protein adsorption processes are comprehensively discussed. The main focus is put on commonly postulated mechanistic aspects and their translation into mathematical concepts and model descriptions. Relevant experimental and computational strategies to practically approach the field of protein adsorption mechanisms and their impact on current successes are outlined.

Mechanism of Membrane Interaction and Disruption by α-Synuclein

Parkinsons disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-β-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.

Supercritical angle fluorescence (SAF) microscopy

We explore a new confocal microscope for the detection of surface-generated fluorescence. The instrument is designed for high resolution imaging as well as for the readout of large biochips. Special feature is the separated collection of two different fluorescence emission modes. One optical path covers the emission into the glass at low surface angles, the other captures high angles, exceeding the critical angle of the water/glass interface. Due to the collection of the supercritical angle fluorescence (SAF) the confocal detection volume is strictly confined to the interface, whereas the low angles collect much deeper from the aqueous analyte solution. Hence the system can deliver information about surfacebound and unbound fraction of fluorescent analyte simultaneously.

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinsons disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

Surface organization and cooperativity during nonspecific protein adsorption events.

Despite many experimental studies on cooperative effects during protein adsorption events, this phenomenon is still poorly characterized and subject of much controversy. In this study, we address the topic of cooperativity using two distinct experimental approaches, namely, kinetic analysis and surface imaging, both based on supercritical angle fluorescence (SAF) microscopy. Several model systems comprising the two proteins BSA and fibrinogen, two different ionic strength conditions and varying pH environments were investigated. The combination of the experimental information obtained from kinetic analysis and from real-time in situ scan images unravel a clear correlation between cooperative adsorption and a heterogeneous protein layer build-up. We propose a mechanistic model of protein adsorption based on an overlap of classical Langmuir-type adsorption on unoccupied surface areas and an additional cooperative adsorption pathway near preadsorbed proteins which is consistent with the experimental observations. Moreover, the growth of two-dimensional surface clusters as an often assumed element of cooperativity could be excluded for the studied systems. The model includes the often observed phenomenon that the adsorption rate decelerates abruptly above a certain coverage limit. Furthermore, the observed evolution of the heterogeneous protein distribution on the surface is in good agreement with the proposed model.

Surface-induced spreading phenomenon of protein clusters

In this study we investigate the behavior of protein clusters on solid surfaces. It is shown that clusters consisting of up to several hundreds of protein monomers can form in solution even at low monomer concentrations and subsequently adsorb to the surface. Using FRET imaging, a significant increase in the inter-protein distance of cluster proteins in time (e.g. a 46% increase after 7 hours on a hydrophobic surface) was revealed indicating that protein clusters spread upon contact with the surface. The spreading rate was strongly dependent on the surface chemistry: fast spreading on hydrophobic, slow spreading on hydrophilic surfaces. Moreover, we found that protein clusters could be deposited on a hydrophobic, OTS-coated surface even though it was already covered with a layer of pre-adsorbed protein monomers. As a result of their high surface mobility these monomers receded from that area to which the protein clusters spread. The molecular rearrangements taking place when a protein cluster deposits onto a surface are discussed in the context of the experimental results.

Understanding cooperative protein adsorption events at the microscopic scale: a comparison between experimental data and Monte Carlo simulations.

Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use a Monte Carlo-type simulation to explore the microscopic details behind cooperative protein adsorption. The simulation was designed to implement our previously proposed mechanism through which proteins are not necessarily rejected if they approach the surface to an occupied region. Instead, we suggest that proteins can be tracked laterally for a certain distance due to the influence of preadsorbed proteins in order to reach the nearest available binding site. The simulation results were compared with experimental data obtained by using the supercritical angle fluorescence (SAF) microscopy technique. It was found that the tracking distance may be up to 2.5 times the proteins diameter depending on the investigated system. The general validity of this tracking mechanism is supported by a number of linear or upward concave adsorption kinetics reported in the literature which are consistent with our simulation results. Furthermore, the self-organization of proteins adsorbing under cooperative conditions on the surface is shown to necessarily cause density inhomogeneities in the surface distribution of proteins which is also in agreement with experimental observations.

Simultaneous near-field and far-field fluorescence microscopy of single molecules.

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

Parallel two-channel near- and far-field fluorescence microscopy

We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the waterglass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.

α-Synuclein Insertion into Supported Lipid Bilayers As Seen by in Situ X-ray Reflectivity

Large aggregates of misfolded α-synuclein inside neuronal cells are the hallmarks of Parkinsons disease. The proteins natural function and its supposed toxicity, however, are believed to be closely related to its interaction with cell and vesicle membranes. Upon this interaction, the protein folds into an α-helical structure and intercalates into the membrane. In this study, we focus on the changes in the lipid bilayer caused by this intrusion. In situ X-ray reflectivity was applied to determine the vertical density structure of the bilayer before and after exposure to α-synuclein. It was found that the α-synuclein insertion, wild type and E57K variant, caused a reduction in bilayer thickness. This effect may be one factor in the membrane pore formation ability of α-synuclein.