Trehalose and calcium exert site-specific effects on calmodulin conformation in amorphous solids.
We have adapted hydrogen/deuterium (H/D) exchange with electrospray ionization mass spectrometry (ESI-MS) to study protein conformation and excipient interactions in lyophilized solids. Using calmodulin (CaM, 17 kD) as a model protein, we demonstrate that trehalose and calcium exert site-specific effects on protein conformation. The effects of calcium are observed primarily in the calcium binding loops, while those of trehalose are observed primarily in non-terminal alpha-helical regions. To our knowledge, this is the first demonstration of site-specificity in the effects of excipients on protein structure in the solid state, and of the utility of H/D exchange with ESI-MS to characterize proteins in amorphous solids.
Li
Yunsong
Y
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA.
Williams
Todd D
TD
Schowen
Richard L
RL
Topp
Elizabeth M
EM
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004339:Drug Compounding / Q000379:methods*; D044367:Phase Transition; D011485:Protein Binding; D011487:Protein Conformation; D014199:Trehalose / Q000737:chemistry*
D002147:Calmodulin; D014199:Trehalose; D002118:Calcium