Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods

Abstract

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.

Figure 1

Ribbon diagram of H124L SNase based on the crystal structure. In each of the single-Trp variants of WT* SNase (P47G/P117G/H124L), Trp140 is replaced by His and the following residues are replaced by Trp: Ile15, Tyr27, Phe61, Phe76, Trp91, Ala102 and His121. The five β-strands (I – V) and three α-helices (H1 – H3) are labeled. The figure was prepared using the program MOLSCRIPT.

Figure 2

Fluorescence spectra of WT* SNase and single-Trp variants at 15°C under native and denaturing conditions. Emission spectra of WT*, Trp76, Trp91, Trp102 and Trp121 SNase with excitation at 280 nm (a) and 295 nm (b) in native buffer (100 mM sodium acetate, pH 5.2); emission spectra of Trp15, Trp27 and Trp61 SNase with excitation at 280 nm (c) and 295 nm (d) in native buffer; emission spectra of WT* and all variants (excitation at 295 nm) in 10 mM phosphoric acid at pH 2.0 (e) and in 100 mM sodium acetate, 2.4 M GuHCl, pH 5.2 (f). All spectra are normalized with respect to an equimolar solution of NATA (shown in gray) in the corresponding buffer.

Figure 3

Refolding kinetic traces of WT* SNase and the seven single-Trp variants induced by a pH-jump from 2.0 to 5.2 at 15°C, measured by the continuous-flow (colored lines) and stopped-flow (circles) methods. Panels (a) and (b) show the kinetics of the proteins with enhanced fluorescence (WT*, Trp76, Tpr91, Trp102 and Trp121 SNase) and quenched fluorescence (Trp15, Trp27 and Trp61 SNase), respectively. Panels (c) and (d) show expanded plot of the sub-millisecond time regime measured by the continuous-flow fluorescence. The solid lines represent the time-courses of folding of these proteins obtained by quantitative kinetic modeling.

Scheme 1