ABSTRACT The characterization of small proteins (<50 amino acids) is an emerging and underexplored field of study. Small proteins are generally thought to be induced by stress and regulate the response to that stress. However, they can also be toxic when overexpressed. Therefore, their levels must be tightly regulated both transcriptionally and posttranslationally. Work in Escherichia coli and Salmonella enterica serovar Typhimurium has revealed small proteins required for growth when magnesium (Mg2+) is limiting. Several of these proteins have been shown to affect the FtsH-dependent degradation of transporters required for the uptake of Mg2+. The molecular details of how small proteins influence the stability of the Mg2+ transporters they interact with remains unknown. Here we aim to understand in molecular detail, using single-particle cryo-electron microscopy (Cryo-EM), how the E. coli small protein MgtS binds the P-type ATPase magnesium transporter MgtA. We hypothesize that the MgtA and MgtS binding interface overlaps with the protease recognition sequence (degron) thereby masking its recognition. To test this hypothesis, structural and in silico models will be used to make mutations that disrupt the predicted binding interface of MgtA and MgtS, and their stabilities will be monitored both in vitro and in vivo. Additionally, we will carry out pulsed stable isotope labeling of amino acids in cell culture (pSILAC) with mass spectrometry-based proteomics during the response and recovery from magnesium limitation or other stress conditions to monitor small protein degradation. Top candidates from our global survey of small protein stability will be tagged and their degradation tested. Co-immunoprecipitation (co-IP) and mass spectrometry will identify potential interacting partners. Finally, we will test which proteases degrade our candidate small proteins and perform degradation assays to observe how the presence or absence of the small protein influences the larger protein’s stability. The results from this proposal will provide critical insights into how small proteins regulate the stability of the larger proteins they bind and identify novel small protein protease adaptors. Given that small proteins are found in the gut microbiota, bacterial pathogens, and in eukaryotes, our approach will serve as a model for studying small protein-big protein interaction and small protein turnover in other model organisms.