L31W as a paradigm two-helix protein
SAP L31W is a suitable system for studying the folding of two-helix bundle proteins. The SAP domain was monomeric at up to millimolar concentrations as determined by NMR measurements of both wild-type and pseudo-wild-type proteins (C.A. Dodson, T.J. Rutherford and J.O.B. Jacobsen, unpublished results). SAP L31W denatured reversibly without aggregation and the equilibrium unfolding transition fitted well within the experimentally accessible window (0–10 M urea, 275–371 K) and multiple repeats of thermal denaturation of both wild-type and pseudo-wild-type SAP domains superimposed (Fig. ). There was also a wide pH range (Fig. A, filled circles) in which the folding and unfolding reactions of SAP L31W were well defined using equilibrium and kinetic biophysical methods.
The tryptophan residue inserted into the core of wild-type SAP domain at position 31 minimally perturbed its structure (Fig. ). The fluorescence reported on the conformational state of the protein. The presence of the tryptophan did not alter its major biophysical properties (the stability of both wild-type and pseudo-wild-type proteins showed the same pH and ionic strength dependencies–Fig. A and B, filled and open circles, respectively).
The chemical denaturation curves of L31W were well defined with good baselines for native and denatured regions under all conditions, apart from the native state at low ionic strength. Addition of NaCl tuned the stability of L31W so that under optimised conditions both native and denatured baselines were well defined (Fig. ). In this way, the ionic strength dependence of the stability of L31W can be exploited, making future Φ-value analysis possible. This is fortunate, since the best mutations for Φ-value analysis are often destabilising and thus have truncated pre-transition baselines compared with the wild-type protein. The acquisition of accurate stability measurements for marginally stable small proteins is complicated as over-truncation impinges on the precision of the thermodynamic parameters determined by curve fitting.
L31W displayed simple folding kinetics
Kinetic transients for L31W were monophasic and the observed rate constants displayed a classic chevron plot with linear arms (Fig. ). Measurement of L31W kinetics on longer timescales or using laser T-jump apparatus (capable of measuring nano-second kinetics) gave a trace with single exponential decays and no evidence of phases faster or slower than those measured here.
Both the kinetic midpoint,
(the [urea] at k50
), and the total m
), determined from unconstrained fits were in agreement with the equilibrium measurements (Table compared with D50
from urea denaturation—parameters in main text above), although the propagated error in
was large, which is inherent in studies on proteins with low values of mu
L31W is a paradigm for two-helix bundle protein folding studies
L31W is one of the simplest naturally occurring protein domains possible and can be considered either as two helices oriented by a connecting loop or a small hydrophobic core being protected from solvent by essentially only a single layer of solvating residues. This minimal composition bridges the divide between studies of isolated elements of secondary structure, which focus on local interactions, and larger globular domains where long-range forces may dominate. L31W is a suitable model protein for extensive studies on folding including Φ-value analysis.