  One of the major problems in the study of the dynamics of proteins is the
visualization of changing conformations that are important for processes
ranging from enzyme catalysis to signaling. A protein exhibiting conformational
dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which
exists in two conformations: the closed (circular) form and the open (linear)
form. It is hypothesized that an increased proportion of the open conformation
leads to the autoimmune disease antiphospholipid syndrome (APS). A
characteristic feature of beta2GPI is the high content of lysine residues.
However, the potential role of lysine in the conformational dynamics of
beta2GPI has been poorly investigated. Here, we report on a strategy to
permanently open up the closed protein conformation by chemical acetylation of
lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific
and complete acetylation was demonstrated by the quantification of primary
amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as
western blot analysis with an anti-acetylated lysine antibody. Our results
demonstrate that acetylated beta2GPI preserves its secondary and tertiary
structures, as shown by circular dichroism spectroscopy. We found that after
lysine acetylation, the majority of proteins are in the open conformation as
revealed by atomic force microscopy high-resolution images. Using this
strategy, we proved that the electrostatic interaction of lysine residues plays
a major role in stabilizing the beta2GPI closed conformation, as confirmed by
lysine charge distribution calculations. We foresee that our approach will be
applied to other lysine-rich proteins (e.g. histones) undergoing conformational
transitions.
