Salivary Proline Rich Protein-Tannins : Intrinsically Unstructured Protein Interaction Model – PROTANIN

1-Scientific background and objectives Physiological roles of saliva are largely related to the salivary proline rich proteins (PRP). PRP contain repeated amino acid sequences and belong to the intrinsically unstructured protein (IUP) family. Among them, basic PRP are involved in precipitation of tannins while glycosylated PRP contribute to mouth lubrication. Tannins are plant polyphenolic compounds that have long been known for their antinutritional properties, as they inhibit digestive enzymes and reduce digestion of proteins and absorption of minerals. Thus, production of PRP is an adaptation process against tannin-rich diets. Moreover, PRP-tannin interactions are responsible for astringency perception that may take part in defense mechanisms against such diets. More rencently, tannins have been attracting much interest for their potential role in the prevention of chronic diseases. This may also be modulated by their interaction with PRP. Owing to their importance for taste and nutritional quality of plant-based foods, interactions between tannins and proteins have been much investigated. However, most of these studies have been performed on peptides that lack the ability to acquire secondary structures through interaction or on proteins that may be irrelevant models of salivary PRP. More generally, studies on IUP are still scarce as their functional roles have only recently been established. Recent development of spectroscopic methods such as nuclear magnetic resonnance (NMR) and small angle X-ray diffusion (SAXS) now make it possible to study their structural and dynamic characteristics in solution. Our objective is to characterize the structure of a human salivary PRP and conformational changes induced by their interactions with tannins. 2-Description of the project, methodology We shall study model systems consisting in, on one hand, human salivary PRP, i.e. basic PRP IB5 and glycosylated PRP II-1, both arising from cleavage of the human salivary pro-protein PRB4S, on the other hand, epigallocatechin gallate (EGCG), the major tannin of green tea, and related structures representing the structural variability of tannins encountered in plant-derived foods. IB5 and II-1 have been obtained by heterologous expression by introducing the gene PRB4S in the genome of the methyltrophic yeast Pichia pastoris. They were secreted into the P. pastoris culture medium and purified. The recombinant approach will also allow production of 15N labeled and doubly labelled 15N,13C PRP to facilitate attribution of NMR signals. Deglycosylated II-1 will also be produced by enzymatic degradation to study the influence of primary sequence and glycosylation. A combination of approaches will be used to characterize first the proteins alone, then the protein-tannin complexes and aggregates and obtain complementary information at various scales. The structural propensities and dynamics of proteins IB5, II-1, and deglycosylated II-1, alone as well as in interaction with various tannins will be analysed by NMR. Electrospray ionisation mass spectrometry (ESI-MS) will be used to determine the stoechiometries of the complexes and, through fragmentation and isotope exchange techniques, gain futher insight on the protein and complex structure. SAXS will enable to obtain basic structural data (overall mass, size and shape of PRP and aggregates, number of proteins and tannins per aggregates) at high resolution. These data will be supplemented by the NMR measurements of interatomic distances and diffusion coefficients. Results concerning the determination of the binding sites of tannins will also be supplemented by the NMR and MS data. Finally, molecular modelling methods are now routinely used to investigate protein folding and conformational changes associated with interactions but are seldom applied to systems involving IUP. The availability of structural data arising in particular from NMR and SAXS experiments will make it possible to develop this new research field. 3-Expected results: We expect to deepen our understanding of (i) the structure and conformation of PRP in solution (ii) the influence of tannins on PRP conformation and folding (iii) the aggregation kinetics and structure of resulting tannin-protein complexes This data will enable better understanding of the molecular mechanisms by which human PRP can bind different tannins and control their biological effects and of the function of salivary PRP. This understanding will also help us to develop a molecular basis for the phenomena that cause astringency in the mouth. It will thus provide tools to control the nutritional and organoleptic qualities of tannin-rich foods and beverages. Beyond the information gained on this particular system, the set of experiments proposed in this project can serve as a model to understand the principles that rule the structure–function relationships of IUP and develop suitable methodologies to investigate such systems.