Characterization of the Unfolding Pathway of Hen Egg White Lysozyme

Abstract

After the recent discovery of a ribonuclease A unfolding intermediate Nature 375, 513-515], we investigated the unfolding pathway of hen egg white lysozyme. At pH* 4.00 with D 2 O at 10°C and 6 M guanidinium chloride, unfolding shows a single, slow kinetic phase, with a relaxation time of 3300 s when monitored by circular dichroism (CD). Exchange of the tryptophan indole nitrogen protons shows that buried Trp residues 123, 111, and 108 lose tight packing and become solvent-exposed simultaneously, with a mean relaxation time of 3300 s, similar to the CDmonitored unfolding rate. Unfolding monitored by Trp fluorescence shows, moreover, that 90% of the amplitude change occurs in a slow phase, with a relaxation time of 2400 s. Faster-unfolding phases with minor amplitudes are detected by Trp indole hydrogen exchange and by fluorescence. It is likely that these changes are caused by Trp 62 and Trp 63, active site residues which are not buried in the hydrophobic core. Lysozyme unfolding was further monitored by the histidine 15 C 1 proton, which gives resolved lines for the native and unfolded species in one-dimensional 1 H-NMR spectra. The majority of the unfolding reaction, 70%, occurs in a slow phase with a relaxation time of 3600 s, but there is also a rapid unfolding phase; 30% of the His 15 C 1 proton resonance intensity is found at the unfolded chemical shift within tens of seconds after the start of unfolding. The amplitude of the rapid unfolding phase increases proportionally with the concentration of GdmCl denaturant present. These results show that a partially buried residue of lysozyme, histidine 15, takes part in forming an unfolding intermediate similar A. 92, 9318-9322]; the difference between that study and ours may reside in the greater sensitivity of 19 F to the detection of motional differences.Kinetic studies of the unfolding reactions of globular proteins monitored by CD 1 or fluorescence show that proteins typically unfold in a single kinetic step, N f U, with no detectable intermediates (Schmid, 1992; Mücke & Schmid, 1994). Thus, it was surprising when Kiefhaber et al. (1995) These results are startling. To test the generality of this behavior, we decided to study the unfolding of hen egg white lysozyme. Hen egg white lysozyme is composed of two domains: an R-domain that contains several helices and a -domain comprised largely of -strands (Figure 1). Lysozyme has many desirable characteristics for this work; its NMR assignments are known (Redfield & Dobson, 1988), and its refolding behavior has been studied in depth (Kiefhaber, 1995;Itzhaki et al., 1994;Dobson et al., 1994). Lysozyme unfolds very slowly, making the study of unfolding by 1D 1 H-NMR practical. Five of the protein's six tryptophan indole nitrogen protons give unique lines in 1D 1 H-NMR spectra. Upon denaturation, these lines disappear and the indole protons resonate in a common line at 10.06 ppm. Moreover, when the Trp side chain is exposed to D 2 O, the indole proton is exchanged for a deuteron and the NMR line disapp...

Real-Time Protein Unfolding: A Method for Determining the Kinetics of Native Protein Denaturation using a Quantitative Real-Time Thermocycler

“…These values gave a sigmoidal plot that was analyzed using the Hill equation (Figure 2B and 3B) (12). Interestingly, we have found that our plots of unfolding rates for HEWL are similar to those by Laurents and Baldwin, whose study used tryptophan fluorescence; however, comparable rates of unfolding could not be obtained (13). …”

Structural and functional analysis of lysozyme after treatment with dielectric barrier discharge plasma and atmospheric pressure plasma jet

“…These results were also supported by a previous study of Takai et al ., who treated lysozyme with He/O 2 gas low-frequency plasma and observed fluorescence quenching. As suggested by a previous study 30 , the majority of the fluorescence emission was due to Trp62 and Trp108 in the native lysozyme structure, with a small contribution to the fluorescence spectra from residues other than Trp that were located near methionine sulphurs or cysteine 39 .…”

Unfolding and Conformational Distributions during Protein Precipitation

“…Given the NMR results, this behavior would be most consistent with the dominant unfolding event being a cooperative unfolding of the -domain, perhaps involving some R-domain residues beyond the core of NMR reporters we examined. Previous investigators have found that the R-domain of lysozyme folds first (Dobson et al, 1994), is more kinetically stable to urea denaturation (Laurents and Baldwin, 1997), and is more thermodynamically stable in 50% trifluoroethanol (Buck et al, 1993). Further, we have found that the R-domain is more stable upon exposure to hydrophobic reversed-phase chromatography surfaces (McNay and Fernandez, 1999).…”