Reaction of hen egg white lysozyme with Fischer-type metallocarbene complexes. Characterization of the conjugates and determination of the metal complex binding sites.
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)5W=C(OR1)R2 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)5W=C(OMe)Me.
Salmain
M
M
Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de Chimie et Biochimie des Complexes Moléculaires (UMR CNRS 7576), Paris, France.
Blais
J C
JC
Tran-Huy
H
H
Compain
C
C
Jaouen
G
G
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004527:Egg Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006838:Hydrocarbons; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D004527:Egg Proteins; D006838:Hydrocarbons; D009942:Organometallic Compounds; C030011:carbene; D009113:Muramidase; D014357:Trypsin; D008697:Methane