Probing residue-level unfolding during lysozyme precipitation.
We have employed nuclear magnetic resonance (NMR) measurements of hydrogen exchange to identify residue-level conformational changes in hen egg white lysozyme (HEWL) as induced by salt precipitation. Deuterated HEWL was dissolved into a phosphate (H2O) buffer and precipitated at pH 2.1 upon addition of solid KSCN or (ND4)2SO4, allowing isotope labeling of unfolded regions. After 1 h, each precipitate was then dissolved at pH 3.8 to initiate refolding and preserve labeling and subsequently purified for NMR analysis. HEWL precipitated by 1.0 M KSCN exhibited increased hydrogen exchange at 14 residues out of 42 normally well-protected in the native state. Of the affected residues, 9 were situated in the beta-sheet/loop domain. A similar, though less extensive, effect was observed at 0.2 M KSCN. Precipitation by 1.2 M (ND4)2SO4 resulted in none of the changes detected with KSCN. The popularity of ammonium sulfate as a precipitant is thus supported by this observed preservation of structural integrity. KSCN, in comparison, produced partial unfolding of specific regions in HEWL due most likely to known preferential interactions between -SCN and proteins. The severity of unfolding increased with KSCN concentration such that, at 1.0 M KSCN, almost the entire beta-sheet/loop domain of HEWL was disrupted. Even so, a portion of the HEWL core encompassed by three alpha-helices remained intact, possibly facilitating precipitate dissolution.
Chang
S T
ST
Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22903-2442, USA.
Fernandez
E J
EJ
D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel / Q000379:methods; D004529:Egg White; D005612:Freeze Drying / Q000379:methods; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014462:Ultrafiltration / Q000379:methods
D009113:Muramidase