Quantitative Assessment of Conformational Heterogeneity in Apolipoprotein E4 Using Hydrogen–Deuterium Exchange Mass Spectrometry

Apolipoprotein E4 (apoE4) is the strongest genetic risk factor for Alzheimer's disease (AD). However, structural differences between apoE4 and the AD-neutral isoform, apoE3, still remain unclear. Recent studies suggest that apoE4 harbors intermediates. However, the biophysical properties and isoform specificity of these intermediates are not known. Here, we use the kinetics of hydrogen-deuterium exchange by mass spectrometry (HDX-MS) to examine the conformational heterogeneities in apoE3 and apoE4. First, we use numerical simulations to compute the HDX-mass spectra of a protein following mixed EX1/EX2 kinetics. The results indicate that in the presence of EX1 kinetics, which is an indicator of conformational heterogeneity, time evolution of the standard deviation (σ(<i>t</i>)) of the spectra exhibits a clear peak, which is dependent on the number of residues (<i>N</i>_EX1) and the rate constant of EX1 kinetics (<i>k</i>_EX1). Then, we performed experiments with several variants of the apoE proteins and compared them with simulation to estimate <i>N</i>_EX1 and <i>k</i>_EX1. Kinetics of the mean deuteration is found to be faster for apoE4, consistent with its poorer stability than apoE3. Importantly, in the case of apoE4, σ(<i>t</i>) exhibits a clear peak at <i>t</i> ∼ 60 s, but apoE3 shows only a small peak at 1800 s. Therefore, both <i>N</i>_EX1 and <i>k</i>_EX1 are larger for apoE4, indicating greater conformational heterogeneity. Notably, the partial EX1 kinetics is observed in both the isolated N-terminal fragment and the full-length form of apoE4, although it is more pronounced in the full-length protein. Moreover, it is enhanced at higher pH and in the presence of bis-ANS. Mutations such as R61T and R112I diminish the EX1 kinetics, making apoE4 behave more like apoE3. Thus, the amino acid substitution at position 112 alters the structural dynamics of the N-terminal domain of apoE4; the changes are further propagated and amplified in the full-length protein. We conclude that HDX-MS is a label-free and robust methodology to characterize structural heterogeneities of proteins even under native conditions. This opens opportunities for screening of the "structure corrector" drug molecules that could convert apoE4 to apoE3-like.