Abstract
Mechanisms of amyloidogenesis are not well understood, including potential structural contributions of mutations in the process. Our previous research indicated that the dimer interface of amyloidogenic immunoglobulin light chain protein AL-09 is twisted 90 degrees relative to the protein from its germline sequence, kappaI O18/O8. Here we report a systematic restoration of AL-09 to its germline sequence by mutating the non-conservative somatic mutations located in the light chain dimer interface. Among these mutants, we find a correlation between increased thermodynamic stability and an increase in the lag time for fibril formation. The restorative mutant AL-09 H87Y completes the trifecta and restores the dimer interface observed in kappaI O18/O8, emphasizing the potential importance of the structural integrity of these proteins to protect against amyloidogenicity. We also find that adding amyloidogenic mutations into the germline protein illustrates mutational cooperativity in promoting amyloidogenesis.
Highlights
Amyloid diseases are characterized by the misfolding of a precursor protein that leads to amyloid fibril formation
We explore the link between thermodynamic stability and fibril formation kinetics and use crystallography to test whether mutants that restore thermodynamic stability restore the canonical dimer interface to the
For the AL-09 restorative mutants, in which individual dimer interface residues are restored to those found in I O18/O8, the most striking result comes from restoring the histidine to tyrosine at position 87 (H87Y)
Summary
Site-directed Mutagenesis—The restorative and reciprocal AL-09 mutants were generated by using the QuikChange Multi Site-directed Mutagenesis kit (Stratagene). Thermal denaturation experiments followed the ellipticity at 218 nm over a temperature range of 4 –90 °C and were analyzed as described previously [12] to calculate a Tm (melting temperature, where 50% of the protein is unfolded). Each sample was equilibrated for 10 min at each urea concentration and the denaturation experiment was followed by CD, acquiring ellipticity at 218 nm for 60 s or by tryptophan fluorescence, with excitation at 294 nm and an emission scan from 310 – 400 nm. Fibril formation kinetics were followed (with each protein in triplicate in a 96-well plate) by measuring ThT fluorescence on a plate reader (Analyst AD, Molecular Devices) with an excitation wavelength of 430 nm and an emission wavelength of 485 nm. Programs REFMAC5 [18] and COOT [19]
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