Structural features of interferon-gamma aggregation revealed by hydrogen exchange.
Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein concentrations to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-gamma was greatly reduced and much less protection from HX in solution was observed. Exchange under these conditions was slower in helix C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to those formed in GdnHCl, the solution phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the native state. Varying the aggregation time before performing HX revealed that KSCN first precipitated native protein and then facilitated partial unfolding of the precipitated protein. These results show that helix C, which forms the hydrophobic core of the IFN-gamma dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the rest of the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state HX patterns may presage regions of the protein susceptible to unfolding during aggregation.
Tobler
Scott A
SA
Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904-4741, USA.
Fernandez
Erik J
EJ
D003903:Deuterium; D019281:Dimerization; D006859:Hydrogen; D007371:Interferon-gamma / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000494:pharmacology; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization
D011994:Recombinant Proteins; D012492:Salts; D012996:Solutions; D006859:Hydrogen; D007371:Interferon-gamma; D003903:Deuterium