Lanthanide interactions with proteins

The E-F hand proteins parvalbumin, troponin C, calmodulin, calbindin and onco-modulin have been studied with respect to lanthanide interactions. The crystal structure of parvalbumin [82] consists of six a-helical regions labeled as A-F. One Ca2+ ion is bound in the loop joining C and D helices and the second ion in the loop of E-F. Calcium ion is bound to six protein ligands in the CD site. Ca2+ in the EF site is bound to seven protein ligands and a water molecule. 

The first protein structures were derived using a technique called isomorphous replacement (IR), developed in the late 1950 s. The materials used are heavy metal derivatives of protein crystals. To obtain a heavy metal derivative of a protein, the protein crystal is soaked in a solution of a heavy metal salt. The metals most used are Pt, Hg, U, lanthanides, Au, Pb, Ag and Ir. The heavy metal or a small molecule containing the heavy metal, depending upon the conditions used, diffuses into the crystal via channels created by the disordered solvent present. The aim is for the heavy metal to interact with some surface atoms on the protein, without altering the protein structure. This is never exactly achieved, but in suitable cases, the changes in structure are slight. 

Interactions of Lanthanides and Their Complexes with Proteins. Conclusions Regarding Magnetic Resonance Imaging... 

On account of the interest in radiolanthanides in tumor scintigraphy, Schomacker et al. [81] studied their interactions with serum proteins and concluded that serum albumin binding predominated upon injection of lanthanide citrate complexes into the bloodstream. The association constant, K, for the equilibrium... 

The physiological result of the binding of the non-essential lanthanide and actinide metals to albumin, or transferrin, in the blood plasma may well be that the metals are held in a form in which they are virtually unable to penetrate the cell membrane in ionic form thus limiting cellular uptake and also the ability to cause harmful effects by interaction with essential enzymes or other proteins. Thus for the non-essential f elements, protein binding may be regarded as part of a protective mechanism against chemical toxicity,... 

Studies with various actinides, following their injection into experimental animals, have shown that after entry into the cells of liver, spleen, kidney and testes, and probably also in other tissues, the metals interact with the subcellular organelles and with the intracellular proteins (Duffield and Taylor 1986). Immediately after entry into the cell the actinides react first with the cytosolic proteins and are then transferred into the membrane-bounded lysosomes within the cell. The rate of transfer to the lysosomes is fastest for americium and curium and slowest for plutonium. In rat or hamster liver plutonium is associated first with an unidentified protein - Protein X -with a molecular mass of 150 (hamster) and 200 kDa (rat) (Neu-Mueller 1988), and then subsequently with the iron-storage protein ferritin. In contrast, americium and curium are rapidly bound to ferritin, without any apparent binding to protein X. Within the lysosomes ferritin appears to be the important binding species (Duffield and Taylor 1986). Much less is known concerning the subcellular distribution of lanthanides, but comparative studies using and Np, Pu and Am confirm the importance of the lysosomes as a deposition site for lanthanides and actinides (Seidel et al. 1986). 

In vivo studies of the reactions of f elements with other specific intracellular proteins do not appear to have been reported, but extensive in vitro investigation of the binding, and the effects of binding, of lanthanides to enzymes and other proteins have been published. Not surprisingly there has been little interest in the study of actinide interactions with such proteins. 

In the application of FRET to detect and quantify protein-target interactions, the protein and the target are each labeled with either a donor or an acceptor prior to being brought into contact. The probes can be conjugated to the macromolecule via primary amine or sulfhydryl or other appropriate chemistries. In general, the unattached probes should be carefully removed by dialysis or other means of buffer exchange. This requirement can be eased when a chelated lanthanide ion is used as the donor. 

Time-resolved RET is capable of very sensitive detection of DNA hybridization. With a lanthanide chelate as the donor and an organic fluorophore like tetramethylrhodamin as the acceptor, time-resolved measurements can indicate the hybridization by strong changes in the intensity decay of the donor [186]. The development of new dyes for time-resolved RET with improved properties still is a major task [187,188]. But, so far, the detection of biomolecular interactions by time-resolved RET has not entered real applications in the DNA or protein array market. 

In order to characterize the active site structure of Ca ATPase from sarcoplasmic reticulum, we have employed Gd + as a paramagnetic probe of this system in a series of NMR and EPR investigations. Gadolinium and several other lanthanide ions have been used in recent years to characterize Ca + (and in some cases Mg2+) binding sites on proteins and enzymes using a variety of techniques, including water proton nuclear relaxation rate measurements (35,36,37), fluorescence (38) and electron spin resonance (39). In particular Dwek and Richards (35) as well as Cottam and his coworkers (36,37) have employed a series of nuclear relaxation measurements of both metal-bound water protons and substrate nuclei to characterize the interaction of Gd + with several enzyme systems. 

Protein biosynthesis occurs on ribosomes which are small organelles composed of proteins and ribosomal RNA (tRNA). Fluorescence and dialysis techniques have been used to study the interaction of Tb3+ with tRNA. The relative fluorescence was found to be proportional to the amount of bound Tb3+ ion. Intact ribosomes produced similar effects on those of tRNA indicating the primary sphere for lanthanide binding is that of ribosomes. 

The application of LSR to amino-acids has received some attention. (451-456, 498) Such studies are an essential preliminary to the use of LSR for amino-acid sequence determination in simple peptides and proteins. The latter are discussed more comprehensively in Section G. A detailed study has been made (453) of the interaction of Eu(iii), Pr(iii), Gd(iii), and La(iii) with iV-acetyl-L-3-nitrotyrosine in order to characterize the nitrotyrosine residue as a potential specific lanthanide binding site in proteins. The parameters of the dipolar interaction indicate a significant contribution from non axially symmetric terms. The conformations of the nucleotides cyclic j8-adenosine 3, 5 -phosphate (3, 5 -AMP) (457, 458) and adenosine triphosphate (ATP) (459) have been deduced using LSR. In the former case the conformation of the ribose and phosphate groups is consistent with the solid state structure. A combination of lanthanide shift and relaxation reagents was used to deduce the most favoured family of conformations for ATP in aqueous solution. One of these conformations corresponds closely to one of the crystal structure forms. 

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