Chemical Modification of Recombinant HIV-1 Capsid Protein p24 Leads to the Release of a Hidden Epitope Prior to Changes of the Overall Folding of the Protein

Abstract

It was found that the affinity of a monoclonal antibody directed against a recombinantly expressed HIV-1 capsid protein p24 (rp24) strongly increased after chemical modification of the Iysine residues of rp24 with different amounts of maleic anhydride. The extent and the sites of modification were analyzed by MALDI-TOF mass spectrometry. Unmodified rp24 and the differently modified rp24 samples were tested for binding the murine monoclonal antibody CB4-1 which recognizes the epitope GATPQDLNTML comprising residues 46-56 of rp24. An increase in the number of modified lysine residues led to enhanced binding affinity of CB4-1. Most pronounced effects were observed after substitution of the first amino groups: an average number of three modified residues per protein molecule increases the binding affinity by a factor of 23, but the substitution of the remaining nine residues increases the binding affinity only by a factor of 11. Fully modified rp24 variant proteins were bound by CB4-1 with Kd values comparable to that of the peptide epitope. Conformation and stability of the unmodified rp24, highly (rp24F, 9 residues; rp24G, 11 residues) modified, and fully modified protein (rp24I, 11 lysine residues and N-terminus) were analyzed by circular dichroism (CD) and fluorescence spectroscopy under different solvent conditions. Little difference in conformation and unfolding behavior was observed between the unmodified and highly modified rp24, which differ drastically in the antibody binding behavior. The fully modified sample, however, displayed a significant decrease in alpha-helical content. Thus, the epitope seems to be hidden (cryptotope) in the unmodified rp24 in a low-affinity binding conformation and becomes displayed at low levels of chemical modification which obviously induce subtle structural changes prior to changes of the overall folding observable by spectroscopic means.

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

“…Other than acetic and succinic anhydrides, the acylation of Lys can also be done with other anhydrides, including citraconic anhydride, 579,580 maleic anhydride, 581,582 trimellitic anhydrides, 583 phthalic anhydride, 584 3-hydroxyl phthalic anhydride, 583 hexahydrophthalic anhydride, 585,586 methyltetrahydrophthalic anhydride, 585,587 succinic anhydride, 588,589 cisaconitic anhydride, 588,590 and fatty acid anhydrides. 591 Unlike succinic anhydride that reverses the charge of Lys upon labeling, methyl acetimidate labeling retains the positive charge on the Lys residue at physiologically relevant pH (Scheme 8, bottom-left).…”

“…Other than acetic and succinic anhydrides, the acylation of Lys can also be done with other anhydrides, including citraconic anhydride, , maleic anhydride, , trimellitic anhydrides, phthalic anhydride, 3-hydroxyl phthalic anhydride, hexahydrophthalic anhydride, , methyltetrahydrophthalic anhydride, , succinic anhydride, , cis -aconitic anhydride, , and fatty acid anhydrides …”

Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

“…Several examples, included chronologically below, are summarized to demonstrate the scope of the protein systems that have been studied with lysine covalent labeling. Welfle and co-workers used maleic anhydride to determine the relative reactivities of lysine residues in the HIV-1 capsid protein p24 (Ehrhard et al, 1996). Results from the labeling experiments along with antibody binding, conformation, and stability assays were used to conclude that an epitope with low-affinity to antibodies is hidden within the p24 and becomes exposed upon conformational changes that were induced by the low levels of maleic anhydride modification.…”

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

“…[12][13][14][15][16][17][18][19][20] Because protein conformational changes affect their surface topology, amino acid residues involved in the structural changes can be identified from differential modification patterns. Information from surface mapping experiments is reliable, though, only if the structural integrity of a protein is preserved during the reaction.…”