Surface topology of Minibody by selective chemical modifications and mass spectrometry

Abstract

The surface topology of the Minibody, a small de novo-designed P-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values.The results of this study indicate that, while overall the Minibody model is correct, the P-sheet formed by the N-and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini s, Sollazzo M, Tramontano A, Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5902-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent

“…structural mapping to large peptides rather than single amino acids (31). On the other hand, probing protein surface topology studies with NHS-biotin and mass spectrometry have been reported (32)(33)(34), but this method, to our knowledge, has not been extended for fine mapping protein-nucleic acid contacts.…”

Assessment of solvent residues accessibility using three Sulfo‐NHS‐biotin reagents in parallel: application to footprint changes of a methyltransferase upon binding its substrate

“…The peptide is detected with only one modification at the lowest ratio, although it contains two possible biotinylatable serine residues and does not encompass any biotinylatable lysine residue. The labeling of both Ser 4 and Ser 14 residues is confirmed by the detection of six different species of modified peptide at higher ratios (Table 2), corresponding to either one or two labels on peptides that can be oxidized. Remarkably, in the case of the monocharged peptide at m/z 2260.004 (ratio 26 : 1, Sulfo-NHS-biotin) reported in Table 2, an alternative attribution as peptide [84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101] where Lys 90 would have been labeled (mass: 81 ppm) does exist.…”

“…Figure 3 shows the product ion mass spectrum recorded by MALDI-TOF-TOF mass spectrometry for this monocharged peptide at m/z 2260.004. This entity corresponds to a mixture of peptides with dehydrated Ser 4 and peptides with dehydrated Ser 14 .…”

Exploitation of proteomics strategies in protein structure-function studies

“…In order to finely tune the specified reaction to this end, an array of experiments can be monitored by electro-spray (ES)-MS [2]. In contrast to the previous classical approach of protein chemistry, one can now make use of both specific and aspecific reagents, and proteases can be used without taking into consideration the total extent of the reaction [3].…”