Development of a Pulse Proteolysis Assay to Investigate Stability in the Measles Polymerase Nucleocapsid Binding Domain
PublisherThe University of Arizona.
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AbstractThe nucleocapsid-binding domains (NBDs) form part of the paramyxovirus replication complex and mediate attachment to the nucleocapsid, a nucleoprotein complex containing the viral RNA genome. NBDs form a very simple helical bundle, and vary between intrinsically unstructured and highly stable in the case of mumps and measles respectively. Despite the differences in properties, these domains show similar sequence and structure. The differences between mumps and measles NBDs provide a point of comparison to investigate differences in structural stability. Using the measles protein as a starting point, we created variants of different stabilities and devised a convenient assay based on pulse proteolysis to rank the variants. A library of NBD variants fused to the SUMO protein were generated by random mutagenesis, and used as a test case for assay development. Three variants of different stabilities were sequenced and their apparent stability differences rationalized based on the mutations present. Pulse proteolysis was then used to screen for the stability and it was concluded to be an effective tool in screening the library for appropriate mutations to be analyzed.
Degree ProgramHonors College
Chemistry & Biochemistry