Protein Folding and Binding Characterized by Mass Spectrometry

Abstract

This presentation will illustrate how the combination of solution phase labeling with mass spectrometry (MS) can elucidate mechanistic aspects of protein behavior. We will focus on two ongoing projects in our laboratory. (A) The initial (submillisecond) stages of protein folding represent a formidable experimental challenge. We have begun to address this issue by using submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for these measurements. Exposure of the protein to a brief pulse of hydroxyl radical (•OH) at different time points during folding introduces covalent modifications at solvent accessible side chains. The extent of labeling is monitored using MS-based peptide mapping, providing spatially-resolved measurements of changes in solvent accessibility. The technique introduced here is capable of providing in-depth structural information on time scales that have thus far been dominated by low resolution spectroscopic probes. (B) The bacterial protease ClpP is a multi-subunit complex with a central degradation chamber that can be accessed via axial pores. In free ClpP these pores are obstructed. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind ClpP and cause the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. We use hydrogen/deuterium exchange MS to obtain insights into the ClpP behavior with and without ADEP1. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore.