Familial Alzheimer’s disease (FAD) is caused by dominant mutations in the amyloid-β (Aβ) precursor protein (APP) and presenilin-1 and -2 (PSEN1, PSEN2). APP is cleaved by β-secretase, then within its single transmembrane domain (TMD) by γ-secretase to produce Aβ, which deposits as cerebral plaques. PSEN is the catalytic component of γ-secretase, a membrane-embedded protease complex. Thus, FAD mutations are only in the substrate and protease that produce Aβ; nevertheless, pathogenic mechanisms remain controversial. Most PSEN FAD mutations show reduced proteolysis (loss of function) but also increase proportions of aggregation-prone 42-residue Aβ peptide (Aβ42) (gain of function). However, γ-secretase has multiple proteolytic functions: Initial endoproteolytic (ε) cleavage of APP substrate produces long Aβ that is trimmed via a carboxypeptidase activity, and FAD-mutant γ-secretases are deficient in this trimming function. New understanding of multiple proteolytic functions of γ-secretase along with recent cryo-EM structure elucidation of the protease-substrate complex now make possible a deeper understanding of effects of FAD mutations. The goal here is to combine chemical, structural, and computational biology to elucidate how presenilin FAD mutations alter γ-secretase structure, dynamics, and function. Such understanding should give insight into how this membrane-embedded protease complex recognizes and processively proteolyzes transmembrane substrates, provide critical clues to pathogenic mechanisms of FAD, and suggest new strategies for prevention of FAD. To this end, we propose to: (1) Develop chemical probes to trap γ-secretase in different stages of substrate interaction for structural analysis by cryo-EM. We developed full TMD substrate mimics to trap active enzyme in a conformation poised for intramembrane proteolysis. Designed variations should allow visualization of the transition states for ε proteolysis, carboxypeptidase cleavage, TMD helix unwinding, and lateral gating of substrate. (2) Perform molecular dynamics (MD) simulations of substrate interaction with FAD-mutant γ-secretase. We computationally restored catalytic aspartates, modeled entry of water to the active site, and captured activation of the computationally restored WT enzyme. We will determine effects of FAD PSEN1 mutations on γ-secretase structural dynamics and interaction with APP substrate and TMD mimics. (3) Develop synthetic substrate probes for analysis of proteolytic dysfunction of FAD-mutant γ-secretase. We developed a set of such functional probes of γ-secretase processing of APP TMD, validating them as convenient and appropriate synthetic surrogates for APP substrate. We will employ these and other proposed substrate probes to determine effects of FAD-mutant γ-secretases on ε proteolysis and specific carboxypeptidase trimming steps.