Premium
Probing residue‐level unfolding during lysozyme precipitation
Author(s) -
Chang Stephen T.,
Fernandez Erik J.
Publication year - 1998
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19980720)59:2<144::aid-bit2>3.0.co;2-h
Subject(s) - chemistry , lysozyme , precipitation , dissolution , salt (chemistry) , residue (chemistry) , crystallography , inorganic chemistry , biochemistry , organic chemistry , physics , meteorology
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 (H 2 O) buffer and precipitated at pH 2.1 upon addition of solid KSCN or (ND 4 ) 2 SO 4 , 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 (ND 4 ) 2 SO 4 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.