Abstract. Protein adsorption on solid surfaces is a widespread phenomenon of large biological and biotechnological significance. Conformational changes are likely to accompany protein adsorption, but are difficult to evidence directly. Nevertheless they have important consequences, since the partial unfolding of protein domains can expose hitherto hidden amino acids. This remodeling of the protein surface can trigger the activation of molecular complexes such as the blood coagulation cascade or the innate immune complement system. In the case of extracellular matrix, it can also change the way cells interact with the material surfaces and result in modified cell behavior. In this review, we present direct and indirect evidences that support the view that some proteins change their conformation upon adsorption. We also show that both physical and chemical methods are needed to study the extent and kinetics of protein conformational changes. In particular, AFM techniques and cryo-electron microscopy provide useful and complementary information. We then review the chemical and topological features of both proteins and material surfaces in relation with protein adsorption. Mutating key amino acids in proteins changes their stability and this is related to material-induced conformational changes, as shown for instance with insulin. In addition, combinatorial methods should provide valuable information about peptide or antibody adsorption on well-defined material surfaces. These techniques could be combined with molecular modeling methods to decipher the rules governing conformational changes associated with protein adsorption.

We have developed a multiwell-based protein aggregation assay to study the kinetics of insulin adsorption and aggregation on hydrophobic surfaces and to investigate the molecular mechanisms involved. Protein-surface interaction progresses in two phases: (1) a lag phase during which proteins adsorb and prefibrillar aggregates form on the material surface and (2) a growth phase during which amyloid fibers form and then are progressively released into solution. We studied the effect of three bacterial chaperones, DnaK, DnaJ, and ClpB, on insulin aggregation kinetics. In the presence of ATP, the simultaneous presence of DnaK, DnaJ, and ClpB allows good protection of insulin against aggregation. In the absence of ATP, DnaK alone is able to prevent insulin aggregation. Furthermore, DnaK binds to insulin adsorbed on hydrophobic surfaces. This process is slowed in the presence of ATP and can be enhanced by the cochaperone DnaJ. The peptide LVEALYL, derived from the insulin B chain, is known to promote fast aggregation in a concentration- and pH-dependent manner in solution. We show that it also shortens the lag phase for insulin aggregation on hydrophobic surfaces. As this peptide is also a known DnaK substrate, our data indicate that the peptide and the chaperone might compete for a common site during the process of insulin aggregation on hydrophobic surfaces.

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The French national project MURE, a collaborative research and development programme, started in March 2014. Two main issues are addressed: coupling of recycling and warm-mix asphalt technique and multi-recycling of bituminous mixtures. It gathers the national road federation, academic laboratories, road contractors and project contracting officials. The objective of the first working group (WG1) of MURE is to validate an accelerated ageing process for bituminous mixtures as well as on site than in laboratory. To achieve this goal, it is first necessary to characterize the ageing using a reliable and reproducible method valid for laboratory and plant-made mixtures. Many studies from the literature showed the efficiency of FTIR spectroscopy for ageing identification. In particular, carbonyl and sulfoxide groups found in binders are appropriate ageing markers. Meanwhile, no common FTIR protocol does exist at present. The first step of the experimental work performed by the WG1 was to compare seven FTIR methods, differing from sample preparation and approaches for calculating spectroscopic indexes. One given bitumen was aged eight times successively by RTFOT method (75 min-163 °C). Two IR spectrum acquisition modes were tested: attenuated total reflectance (ATR) and transmission (spreading of bitumen on an IR transparent plate and bitumen dissolution in a solvent). The second step of the study was to verify the repeatability of FTIR tests on neat and aged bitumen according to sample preparation and calculation methods. The results allow confirming the relevance of FTIR spectroscopy for qualifying bitumen ageing. A single methodology and a common tool for harmonizing the calculation of indexes are proposed. The whole procedure can be used to follow the ageing properties on site at short and long times.

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