Probing residue-level unfolding during lysozyme precipitation

Abstract

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.

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

“…Using HX and two-dimensional NMR, Chang and Fernandez (1998) previously obtained information about protein unfolding during salt-induced precipitation of lysozyme. Native exchange patterns were observed for lysozyme precipitated with ammonium sulfate, a model kosmotropic salt.…”

“…HX in conjunction with NMR has been used extensively in the last two decades for protein stability measurements (Englander and Kallenbach, 1984;Bai et al, 1994) and in folding experiments (Roder et al, 1988;. In addition, unfolding during processes such as lyophilization (Desai et al, 1994), precipitation (Chang and Fernandez, 1998), and reversed-phase chromatography (McNay and Fernandez, 1999) has been investigated using HX and NMR. Further, mass spectrometry (MS) has more recently emerged as a tool to detect HX in proteins (Katta and Chait, 1993;Miranker et al, 1993;Zhang and Smith, 1993;Chung et al, 1997).…”

“…The extent of unfolding, as well as the fraction of protein that remained insoluble upon redissolution, increased with increasing thiocyanate concentration. The insoluble fraction exhibited a more complete exchange, supporting the idea that those fractions represent denatured, aggregated protein (Chang, 1999). While these two-dimensional NMR data provided detailed information about domains and residues involved in unfolding, questions remain about the distributions of conformers present.…”

“…While these two-dimensional NMR data provided detailed information about domains and residues involved in unfolding, questions remain about the distributions of conformers present. For example, NMR results indicated a hydrogen occupancy of about 30% for a set of amide sites after precipitation with 1.0 M KSCN (Chang and Fernandez, 1998). If all of the molecules had unfolded in the same way, but only 30% of the newly exposed sites exchanged due to limiting intrinsic HX rates, then one peak in a mass spectrum would have been observed.…”

“…If all of the molecules had unfolded in the same way, but only 30% of the newly exposed sites exchanged due to limiting intrinsic HX rates, then one peak in a mass spectrum would have been observed. However, electrospray ionization mass spectrometry (ESI-MS) analysis revealed two peaks, one heavier peak that represented molecules that remained relatively native during the process, and a lighter peak representing ones that had unfolded (Chang, 1999).…”