Conformational-Sensitive Fast Photochemical Oxidation of Proteins and Mass Spectrometry Characterize Amyloid Beta 1–42 Aggregation

Preventing and treating Alzheimer’s disease require understanding the aggregation of amyloid beta 1–42 (Aβ_1–42) to give oligomers, protofibrils, and fibrils. Here we describe footprinting of Aβ_1–42 by hydroxyl radical-based fast photochemical oxidation of proteins (FPOP) and mass spectrometry (MS) to monitor the time-course of Aβ_1–42 aggregation. We resolved five distinct stages characterized by two sigmoidal behaviors, showing the time-dependent transitions of monomers-paranuclei-protofibrils-fibrillar aggregates. Kinetic modeling allows deciphering the amounts and interconversion of the dominant Aβ_1–42 species. Moreover, the irreversible footprinting probe provides insights into the kinetics of oligomerization and subsequent fibrillar growth by allowing the conformational changes of Aβ_1–42 at subregional and even amino-acid-residue levels to be revealed. The middle domain of Aβ_1–42 plays a major role in aggregation, whereas the N-terminus retains most of its solvent-accessibility during aggregation, and the hydrophobic C-terminus is involved to an intermediate extent. This approach affords an in situ, real-time monitoring of the solvent accessibility of Aβ_1–42 at various stages of oligomerization, and provides new insights on site-specific aggregation of Aβ_1–42 for a sample state beyond the capabilities of most other biophysical methods

Fast Photochemical Oxidation of Proteins and Mass Spectrometry Follow Submillisecond Protein Folding at the Amino-Acid Level

We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast photochemical oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose “protection” changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix₁ shows a decreasing trend of oxidative modification during the first 0.1–1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix₁ formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen−Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

We describe a platform utilizing two methods based on hydrogen–deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein–ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 μM for the three different regions, giving an overall binding affinity of 7.4 μM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides <i>site-specific</i> binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, Δ<i>C</i>_1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein–ligand interactions

Continuous and Pulsed Hydrogen–Deuterium Exchange and Mass Spectrometry Characterize CsgE Oligomerization

We report the use of hydrogen–deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE