Exploring the mechanism of selective noncovalent adduct protein probing mass spectrometry utilizing site-directed mutagenesis to examine ubiquitin.
Mass spectrometry (MS) is emerging as an additional tool for examining protein structure by way of experiments where structurally related mass changes induced in solution are subsequently detected in the gas phase. Selective noncovalent adduct protein probing (SNAPP) is a recent addition to this type of experiment. SNAPP utilizes noncovalent recognition of lysine residues with 18-crown-6 (18C6) to monitor changes in protein structure. It has been observed that the number of 18C6 adducts that attach to a protein is a function of the structure of the protein. The present work seeks to examine the underlying chemistry which controls the differential attachment of 18C6 to lysine by using ubiquitin as a model system. Ubiquitin is a small protein with a structure that has been well characterized by multiple techniques. Site-directed mutagenesis was used to create a series of ubiquitin mutants where the lysine residues were exchanged for asparagine one at a time. These mutants were then evaluated by SNAPP-MS to determine the relative contribution of each lysine as a binding site for 18C6. It was found that attachment of 18C6 is largely controlled by the strength of intramolecular interactions involving lysine residues. Salt bridges provide the greatest interference, followed by hydrogen bonds. In addition to determining the mechanism for SNAPP, insights are provided about the structure of ubiquitin including confirmation of the existence of two dynamic states for the native structure. These results are discussed in relation to the biological functions of ubiquitin.
Liu
Zhenjiu
Z
Department of Chemistry, University of California, Riverside, California 92521, USA.
Cheng
Shijun
S
Gallie
Daniel R
DR
Julian
Ryan R
RR
D002942:Circular Dichroism; D013058:Mass Spectrometry / Q000379:methods*; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics
D015335:Molecular Probes; D025801:Ubiquitin