Reaction of hen egg white lysozyme with Fischer‐type metallocarbene complexes

Abstract

The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available reagents for particular target residues. On the other hand, transition organometallic chemistry offers an almost untouched field for this purpose. In particular, Fischer-type metallocarbene complexes of the general formula (CO) 5 W¼C(OR 1 )R 2 may be attractive reagents because they contain the heavy element tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein-aminocarbene conjugates that have an average of one aminocarbene moiety per protein molecule. RP-HPLC combined with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analysis of the conjugates revealed that they were mixtures of the native protein, monoaminocarbenes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI-TOF-MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were involved in the reaction of HEWL with (CO) 5 W¼C(OMe)Me.Keywords: Fischer-type metallocarbene complexes; lysine residues; lysozyme; MALDI-TOF-MS.The preparation of heavy metal derivatives, i.e. the incorporation of heavy atoms into protein crystals, is the traditional approach to determine diffraction phases in the course of protein 3D structure resolution by X-ray crystallography [1]. This step is essential for the construction of an electrondensity map and therefore a 3D model of the protein. Suitable reagents should make it possible to incorporate the heavy element at a limited number of binding sites with a high occupancy factor and not disturb the crystal packing or protein folding (isomorphous replacement). Unfortunately, selection of reagents is often a trial-and-error process because such reagents are not selective of particular binding sites [2]. Moreover, mercury compounds are known to be toxic to handle.In the last few years, we have been designing new heavy metal reagents based on transition organometallic complexes. This particular class of metal complexes made it possible to design side-chain-selective reagents yielding covalent protein adducts [3 -5].More recently, we have shown that Fischer-type metallocarbene complexes of the general formula 1 react with a model protein, namely BSA, to form stable conjugates [6]. Indeed, these compounds display a highly electrophilic character at the carbene carbon atom and are therefore susceptible to nucleophilic substitution by amines (aminolysis) to yield very stable metalloaminocarbenes. (Scheme 1).This preliminary result suggested that these complexes may act as heavy atom reagents because (a) they target particular side chains and (b) they form covalent and stable adducts. We describe here the reaction of two Fischer-type carbene complexes, 1a and 1b, with the enzyme hen egg white...

Solution‐ and Crystal‐Phase Covalent Modification of Lysozyme by a Purpose‐Designed Organoruthenium Complex. A MALDI‐TOF MS Study of its Metal Binding Sites

“…Curiously, ruthenocenyl pyridinium peptide (6 ± 14)Ru was detected in two different and well separated fractions. In the course of our previous work on tungsten pentacarbonyl aminocarbene conjugates of lysozyme, similar behaviour was observed for peptide (6 ± 14)W. [19] No satisfactory explanation was found to explain this unusual behaviour. Finally, the 38.8 min retention time peak was assigned to peptide (1 ± 5)Ru; that is, labelling at Lys1.…”

“…Acylation of HEWL with activated ester derivatives of estrone glucoronide [51] and pregnanediol glucoronide [52] also occurred at Lys33, Lys97, together with Lys116. Interestingly, the hydrophobic amine-targeted reagents (CO) 5 W(OMe)Me [19] and dinitrofluorobenzene [53] were also shown to react preferentially with Lys13, Lys33 and Lys116. This indicates that the accessibility of the individual amine functions is not the sole parameter that governs their reactivity.…”

“…On the whole, these mixtures of adducts seemed to be more heterogeneous than those resulting from the reaction of (4-benzene chromium tricarbonyl) pyrylium ions [11] or metallo-carbene complexes. [19] This might indicate that a greater number of protein sites were involved in the reaction with 1. Moreover, under the same elution conditions, the ruthenocenyl pyridinium adduct species were less well separated than the metallo-aminocarbene conjugate species, primarily because the positive charge of the amine-bearing residue after reaction is conserved in the first case whereas the aminocarbene residue is neutral, and therefore more hydrophobic.…”

Biophysical characterisation of adducts formed between anticancer metallodrugs and selected proteins: New insights from X-ray diffraction and mass spectrometry studies

“…Just a few additional structures, somehow connected to our topics, had been obtained by Jaouen and coworkers working on specific lysozyme modifications caused by a variety of organometallic agents [36,37]. In contrast, a quite conspicuous number of crystallographic structures were available in the HAD data bank in relation to the heavy atom replacement methodology [38].…”

Cysteine‐Specific, Covalent Anchoring of Transition Organometallic Complexes to the Protein Papain from Carica papaya

“…However, as proteins generally carry a lot of these functions, labelling was not chemoselective but generally involved several binding sites, which yielded mixtures of modified proteins. For example, treatment of the metallocarbene complex (CO) 5 W=CA C H T U N G T R E N N U N G (OMe)Me with HEWL (in solution) led to the introduction of the heavy atom at four different positions, [28] whereas labelling of HEWL with a ruthenocenyl pyrylium salt led to the introduction of ruthenium at five different position in the crystal state. [29] Very recently, another research group successfully made use of the reactivity of an arene ruthenium(II) complex to regiospecifically coordinate a ruthenium centre to the imidazole side chain of His15 in HEWL.…”