Temperature sensitive (TS) missense mutants have been foundational for characterization of essentialgene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TSmissense mutants within the context of their functional proteomes is lacking. We applied massspectrometry (MS) based thermal proteome profiling (TPP) to investigate the proteome-wide effects ofmissense mutations in an application that we refer to as mutant Thermal Proteome Profiling (mTPP).This study characterized global impacts of temperature sensitivity-inducing missense mutations in twodifferent subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and globalproteomics were similar between the mutants, which could suggest that a similar functional disruption isoccurring in both missense variants. Results from mTPP, however, provide unique insights into themechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were notobtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is aprecise approach to measure changes in missense mutant containing proteomes without the requirementfor large amounts of starting material, specific antibodies against proteins of interest, and/or geneticmanipulation of the biological system. Although experiments were performed under permissiveconditions, mTPP provided insights into the underlying protein stability changes that cause dramaticcellular phenotypes observed at non-permissive temperatures. Overall, mTPP provides uniquemechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in arapid, non-biased fashion.
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