New Insights on Growth Mechanisms of Protein Clusters at Surfaces: an AFM and Simulation Study

Abstract

Despite its relevance to a wide range of technological and fundamental areas, a quantitative understanding of protein surface clustering dynamics is often lacking. In inorganic crystal growth, surface clustering of adatoms is well described by diffusion-aggregation models. In such models, the statistical properties of the aggregate arrays often reveal the molecular scale aggregation processes. We investigate the potential of these theories to reveal hitherto hidden facets of protein clustering by carrying out concomitant observations of lysozyme adsorption onto mica surfaces, using atomic force microscopy, and Monte Carlo simulations of cluster nucleation and growth. We find that lysozyme clusters diffuse across the substrate at a rate that varies inversely with size. This result suggests which molecular scale mechanisms are responsible for the mobility of the proteins on the substrate. In addition the surface diffusion coefficient of the monomer can also be extracted from the comparison between experiments and simulations. While concentrating on a model system of lysozyme-on-mica, this 'proof of concept' study successfully demonstrates the potential of our approach to understand and influence more biomedically applicable protein-substrate couples.

Lysozyme adsorption at a silica surface using simulation and experiment: effects of pH on protein layer structure

“…24 Previous work by Sander et al use the Random Sequential Adsorption model for the monolayer, 39 however this is at odds with AFM images of lysozyme adsorption onto charged mica surfaces which show that close-packing models are more appropriate. 21,22,40 Therefore our best estimate for the protein surface amount of an adsorbed monolayer is 203-288 ng cm À2 . This figure is for the protein only, without entrained solvent.…”

Understanding protein adsorption phenomena at solid surfaces

“…Considering that the observation of increasing adsorption kinetics alone can also be misinterpreted [44], scanning force microscopy (SFM) is undoubtedly the method of choice to experimentally support the idea of two-dimensional cluster formation due to its atomic scale resolution. Numerous further SFM studies in which protein adsorption was directly measured in solution, i.e., without a preceding drying step, could reveal the existence of twodimensional protein surface clusters [149,[182][183][184][185][186]. However, the often proposed cluster growth mechanism through a surface diffusion or a direct attachment of protein monomers can not be observed directly but can only indirectly concluded from the increase of the cluster size.…”

“…In the field of protein adsorption AFM is often applied to image the distribution of proteins within a layer with a special regard to surface aggregation [149,150,[182][183][184][185][186]225]. Even protein adsorption kinetics, albeit at very poor temporal resolution, and conformational changes of individual proteins were measured [226,227].…”

“…One solution is to propose arbitrary interaction energies for the adsorption of a protein, its transition into another state, or its aggregation with other pre-adsorbed proteins [186,297,298,300]. Another possibility consists of replacing approximate models with empirical parameters [149,186,298,299]. Calculating the Boltzmann factor from the energy gain/loss of an MC step serves to determine the probability that this step is accepted.…”

Protein adsorption mechanisms on solid surfaces: lysozyme-on-mica

“…A model system of lysozyme (L6876, Sigma-Aldrich, Brøndby, Denmark) adsorption onto mica has been used to demonstrate this methodology [10,11]. Lysozyme is globular and fairly robust so that reliable observation using in-liquid AFM is viable, and mica provides a flat surface for microscopy while presenting a charged surface to attract the protein.…”