CHARACTERIZATION OF SURFACE-TOPOLOGY AND BINDING AREA IN COMPLEXES OFTHE ELONGATION-FACTOR PROTEINS EF-TS AND EF-TU-CENTER-DOT-GDP FROM THERMUS-THERMOPHILUS - A STUDY BY PROTEIN CHEMICAL MODIFICATION AND MASS-SPECTROMETRY

Amino acetylation of lysine residues in combination with mass-spectrom etric peptide mapping of tryptic peptides has been applied to the char acterization of surface topology and binding areas in complexes of the translation-elongation factors EF-Tu and EF-Ts from Thermus thermophi lus. Electrospray/ionization mass spectrometry (ESI-MS) yielded pH-dep endent differences in the charge structure for the molecular ions of E F-Ts indicating differences in solution structure. The noncovalent EF- Tu.GDP complex was directly identified by ESI-MS. Acetylation reaction s of EF-Ts, EF-Tu.GDP, and EF-Ts EF-Tu complexes with varying molar ra tios of acetic anhydride were directly followed by mass spectrometry, which identified the precise number of acetyl groups in the partially modified proteins. The acetylation sites and relative reactivities of the lysine residues were determined from mass-spectrometric peptide-ma pping data of tryptic peptide mixtures; the modified sites were identi fied by on-line HPLC-ESI-MS. The comparison of the acetylation pattern for the free proteins with that of the EF-Tu.EF-Ts complex enabled th e characterization of structural changes in the effector loop of EF-Tu , around the Lys-45 and Lys-52 residues, upon complex formation. Two d omains in EF-Ts, the N-terminal region comprising Lys-21 and Lys-45 an d a central helix-turn-helix region comprising Lys-119, Lys-133, and L ys-140, were shielded from acylation in the complex; this shows their different accessibilities in free and complexed EF-Ts. These results s how the usefulness of an approach combining of differential chemical m odification combined with mass spectrometry for probing surface topolo gy and molecular interactions in protein complexes.