Protein Crystallography through Supramolecular Interactions between a Lanthanide Complex and Arginine

Abstract

The determination of the complete sequence of the human genome triggered the emergence of structural proteomics, a challenging field of research devoted to the determination of the structure of proteins at atomic resolution to understand their structure-function relationships. Among the various techniques for structure determination, [1] X-ray crystallography plays an essential role. Its bottleneck is the serendipitous and time-consuming preparation of well-diffracting crystals. Once such crystals are obtained, de novo structure determination requires the preparation of at least one heavy-atom derivative. Nowadays, methods based on anomalous diffraction are routinely used.[2] Lanthanide ions are thus very convenient for this purpose because of their large anomalous signal with either synchrotron or Cu Ka radiation. Lanthanide derivative crystals of the protein may be prepared by: 1) replacing Ca 2+ by Ln 3+ ions in calcium-binding proteins, [3] 2) covalent grafting of dysprosium complexes [4] or a terbium binding tag, [5] or 3) cocrystallization with macrocyclic gadolinium, ytterbium, or lutetium complexes.[6] The last method is very easy to carry out, but the interactions between the lanthanide complexes and the protein are difficult to predict. To shed light on this problem, we recently developed a research program devoted to the study of the solid-state and solution interactions between lanthanide complexes and biological macromolecules.Herein we describe the properties of tris(dipicolinate)-lanthanide complexes [Ln(dpa) 3 ] 3À (dpa = dipicolinate = pyridine-2,6-dicarboxylate), [7] which could be highly interesting for macromolecular crystallography, since 1) successful derivatization can easily be detected through the intrinsic luminescence of the europium and terbium complexes, 2) the new crystal form of derivatives with hen egg-white lysozyme (HEWL) evidenced strong interactions between the tris(dipicolinate)lanthanide complexes and the protein; these interactions were shown to involve guanidinium moieties of arginine residues, and 3) the occurrence of these supramolecular interactions ocurred both in aqueous solution as well as in the solid state, with the ethylguanidinium cation (EtGua + ) mimicking the behavior of accessible arginine groups of proteins.Cocrystallized derivative crystals of HEWL were obtained by the hanging-drop method. Mixing HEWL and A 3 [Ln(dpa) 3 ] (Figure 1 a, Ln = Eu, Tb; A = Li, Na, Cs, NH 4 ) led to a precipitate from which crystals grew within a few days. The shape of the resulting crystals (Figure 1 b) was very different from that of native tetragonal crystals obtained under similar conditions.[8] Irradiation with UV light (l ex =

Water‐Soluble Luminescent Hybrid Composites Consisting of Oligosilsesquioxanes and Lanthanide Complexes and their Sensing Ability for Cu2+

“…[14] It has been reported that [Ln(dpa) 3 ] 3À complexes can be fixed on the protein through hydrogen bonds between the oxygen atomso ft he carboxylate groups of dpa and the protein hydrogen-bond donors such as the amino groups. [15] The resulting hybrid composites are highly soluble in water,d isplaying improveds tability and prolonged lifetimei nc omparison with the pure complex, and they also presenthighly selective and sensitive sensing for Cu 2 + .…”

Paramagnetic DOSY: An Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins

“…[7] Finally following pioneering workso fK ahn andc o-workers in the early 2000s, it is possiblet oi ncorporate lanthanide complexesi nto bio-macromolecules via an etwork of non-covalent interactions for X-rays crystallography structure determination. [8] This methodi sv ery simple and easy to perform but is less predictable since the study of lanthanide complexes-protein interactions is an almoste mptyf ield of research [9] In this context,w ed escribed the use of tris-dipicolinate lanthanide complexes ([Ln(DPA) 3 ] 3À ,D PA = 2,6-pyridine dicarboxylate; Figure1)t hat are ablet oc o-crystallize efficiently with al arge number of proteins [10] andw ee videnced ap articulara ffinity of this complexf or cationic amino acids according to the sequence arginine > histidine > lysine. [11] The groups of Otting and Su have also used these complexes to determine the structure of the proteins ArgN andu biquitin in solution by NMR.…”

The prominent role of resonant elastic scattering for solving the X-ray structure of macromolecules

“…However, incorporation of lanthanides by using lanthanide salts often damages protein crystals, owing to the preferred nine-based coordination of lanthanide ions. One solution to overcome this problem is to use lanthanide complexes made of a ligand that surrounds the lanthanide ions as a cage [14,15]. These complexes can be easily introduced in protein crystals and turn out to be efficient vehicles for phasing using anomalous dispersion, in particular for large macromolecular assemblies (for a short overview of protein structures determined using these lanthanide complexes, see [13]).…”