ISSN:
0006-3592
Keywords:
lysozyme
;
protein precipitation
;
thiocyanate
;
hydrogen exchange
;
nuclear magnetic resonance
;
protein unfolding
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
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 β-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 β-sheet/loop domain of HEWL was disrupted. Even so, a portion of the HEWL core encompassed by three α-helices remained intact, possibly facilitating precipitate dissolution. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 144-155, 1998.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
